Compounds for inhibiting the interaction of BCL2 with binding partners

ABSTRACT

The present invention relates to compounds of formula (I) in which R 1 , R 2 , R 3  and R 4  are as defined in the Summary of the Invention. Compounds of formula I are capable of disrupting the BCL-2 interations with proteins containing a BH3 domain. Disrupting this interaction can restore the anti-apoptotic function of BCL-2 in cancer cells and tumor tissue expressing BCL-2. The invention further provides a process for the preparation of compounds of the invention, pharmaceutical preparations comprising such compounds and methods of using such compounds in the treatment of cancerous diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/US2012/069181 filed Dec. 12, 2012 whichclaims the benefit of priority to U.S. provisional patent applicationNo. 61/579,731, filed 23 Dec. 2011. The full disclosures of theseapplications are incorporated herein by reference in their entiretiesand for all purposes.

BACKGROUND

1. Field of the Invention

The present invention relates to compounds capable of disrupting theBCL-2 interations with proteins containing a BH3 domain. Disrupting thisinteraction has the potential to restore the anti-apoptotic function ofBCL-2 in cancer cells and tumor tissue expressing BCL-2. The inventionfurther provides a process for the preparation of compounds of theinvention, pharmaceutical preparations comprising such compounds andmethods of using such compounds in the treatment of cancer.

2. Background of the Invention

Apoptosis, or programmed cell death, is important for normalembryological or anatomical development, host defense and suppression ofoncogenesis. Faulty regulation of apoptosis has been implicated incancers and many other human diseases which result from an imbalancebetween the process of cell division and cell death. BCL-2 belongs to afamily of proteins which regulate apoptosis. BCL-2 contributes to cancercell progression by preventing normal cell turnover caused byphysiological cell-death mechanisms.

The expression levels of BCL-2 proteins correlates with resistance to awide spectrum of chemotherapeutic drugs and γ-radiation therapy.Over-expression of BCL-2 has been observed in many forms of cancer. Thefollowing over-expression percentages in cancers have been observed:20-40% in prostrate; 80-100% in hormone resistant prostrate; 60-80% inbreast; 20-40% in non-small cell lung; 60-80% in small cell lung;50-100% in colorectal; 65% in melanoma; 13% in head and neck; and 23% inpancreatic.

Biological approaches to modulating Bcl-2 function using anti-senseoligonucleotides or single-chain antibodies have been shown to enhancetumor cell chemosensitivity. Synergistic effects and complete tumorregression have been observed in vivo in the combined treatments with acombination of an anti-sense oligonucleotide (G3139) and docetaxel.Therefore, BCL-2 represents a highly attractive target for thedevelopment of a novel therapy for the treatment of many forms ofcancers. In particular, the need exists for small molecules that bind toBCL-2 and block its anti-apoptotic function in cancer and promote celldeath in tumors. The present invention fulfills this need.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds of Formula I:

in which:

R₁ is selected from hydrogen and halo;

R₂ is selected from hydrogen and C₁₋₄alkyl; wherein R₂ is in the metaposition and R₃ is in the para position relative to the pyrazole ring orR₂ is in the para position and R₃ is in the meta position relative tothe pyrazole ring;

R₃ is selected from hydroxy and -L-R₅; wherein L is —NHS(O)₂X₁—; whereinX₁ is selected from a bond and branched or unbranched C₁₋₄alkylene;wherein said alkylene of X₁ can be unsubstituted or substituted with agroup selected from carboxy-methyl, methoxy-carbonyl-methyl,methyl-carbonyl-amino, hydroxy-methyl and phenyl;

R₄ is selected from hydrogen, hydroxy, —X₃NR₈R₉, —X₃C(O)OR₈, —X₃OR₈,—X₃C(O)NR₈R₉ and —X₃NR₈C(O)R₉; wherein X₃ is selected from a bond andC₁₋₄alkylene; and R₈ and R₉ are independently selected from hydrogen,C₁₋₄alkyl and phenyl; or R₈ and R₉ together with the nitrogen to whichR₈ and R₉ are attached form a 5 to 7 member saturated ring containing 1to 3 groups or heteroatoms independently selected from C(O), NR₁₀, O andS(O)₀₋₂; wherein R₁₀ sis selected from hydrogen and C₁₋₄alkyl;

R₅ is selected from hydrogen, C₁₋₆alkyl, C₂₋₆alkenyl, cyclopropyl,imidazo[1,2-a]pyrimidinyl, 2-oxo-4-phenylpiperazin-1-yl,4-(2-chlorobenzyl)-3-oxopiperazin-1-yl, imidazo[1,2-a]pyridinyl,benzo[d]isoxazolyl, naphtho[2,1-d][1,2,3]oxadiazol-5-yl,1H-pyrrolo[2,3-b]pyridinyl, imidazo[2,1-b]thiazolyl,1H-pyrazolo[3,4-b]pyridinyl, benzo[c][1,2,5]thiadiazolyl,4-oxo-4,5,6,7-tetrahydrobenzofuranyl,(7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methyl,benzo[c][1,2,5]oxadiazolyl, 2-oxo-1,2,3,6-tetrahydropyrimidinyl,1,2,4-oxadiazolyl, 2,3-dihydrobenzo[b][1,4]dioxin-2-yl,naphtho[2,3-d][1,3]dioxol-2-yl, 3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl,2,3-dihydrobenzofuran-3-yl, chroman-8-yl, 3-oxo-3H-pyrazolyl,6-oxo-1,6-dihydropyridazinyl, benzo[b]thiophenyl, dimethyl-amino,benzo[b]furanyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl,isoindoline-1,3-dionyl, 2-oxo-1,2-dihydropyridinyl,2-oxo-1,2,5,6,7,8-hexahydroquinolinyl,4-oxo-1,4-dihydro-1,8-naphthyridinyl, 4-oxo-4H-pyrano[2,3-b]pyridinyl,10,10-dioxido-9-oxo-9H-thioxanthen-3-yl, 5-oxopyrrolidin-3-yl, phenyl,quinolinyl, isoquinolinyl, benzyl, phenoxy, phenylthio, benzoxy,phenyl-sulfonyl, furanyl, thiazolyl, oxazolyl, isoxazolyl, thienyl,pyrrolyl, quinolin-8-yloxy, pyrimidinyl, pyridinyl, pyrrolidinyl,pyrrolidinonyl, imidazolidine-2,4-dionyl, piperidinyl, piperazinyl,pyrazinyl, pyrazolyl, morpholino, oxomorpholino, indolyl,benzo[b]thiophenyl, benzo[b]furanyl, benzo[d][1,2,3]triazol andoxopiperazinyl; wherein said C₁₋₆alkyl, C₂₋₆alkenyl, cyclopropyl,imidazo[1,2-a]pyrimidinyl, benzo[d]isoxazolyl, imidazo[1,2-a]pyridinyl,4-oxo-4,5,6,7-tetrahydrobenzofuranyl,2-oxo-1,2,3,6-tetrahydropyrimidinyl, imidazo[2,1-b]thiazolyl,1H-pyrrolo[2,3-b]pyridinyl, 1H-pyrazolo[3,4-b]pyridinyl,1,2,4-oxadiazolyl, benzo[c][1,2,5]thiadiazolyl,2,3-dihydrobenzo[b][1,4]dioxin-2-yl, naphtho[2,3-d][1,3]dioxol-2-yl,3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl, 1,2,5-oxadiazolyl,1,2,4-oxadiazolyl, benzo[c][1,2,5]oxadiazolyl,isoindoline-1,3-dionyl,2,3-dihydrobenzofuran-3-yl, chroman-8-yl,3-oxo-3H-pyrazolyl, 6-oxo-1,6-dihydropyridazinyl,2-oxo-1,2-dihydropyridinyl, 2-oxo-1,2,5,6,7,8-hexahydroquinolinyl,4-oxo-1,4-dihydro-1,8-naphthyridinyl, 4-oxo-4H-pyrano[2,3-b]pyridinyl,10,10-dioxido-9-oxo-9H-thioxanthen-3-yl, 5-oxopyrrolidin-3-yl, phenyl,quinolinyl, isoquinolinyl, phenoxy, benzyl, benzoxy, phenoxy-methyl,phenylthio, phenyl-sulfonyl, furanyl, thiazolyl, oxazolyl, isoxazolyl,thienyl, pyridinyl, pyrrolyl, quinolin-8-yloxy, pyrrolidinyl,pyrimidinyl, pyrrolidinonyl, piperazinyl, piperidinyl, pyrazinyl,pyrazolyl, morpholino, oxomorpholino, indolyl, benzo[d][1,2,3]triazol oroxopiperazinyl of R₅ is unsubstituted or substituted with 1 to 3 groupsindependently selected from halo, cyano, nitro, —NR₆R₇, C₁₋₄ alkyl,halo-substituted-C₁₋₄ alkyl, C₁₋₄ alkoxy, halo-substituted-C₁₋₄ alkoxy,halo-substituted-C₁₋₄ alkylthio, —C(O)OR₆, —X₃OR₆, —C(O)R₆, —C(O)NR₆R₇,—NR₆S(O)₂X₃R₇, —X₃NR₆C(O)R₇, —S(O)₀₋₂R₆, —S(O)₀₋₂NR₆R₇, phenyl, benzyl,oxazolyl, naphthyl, piperidinyl, pyrrolidinyl, morpholino,morpholino-methyl, 1,2,4-oxadiazolyl, pyrazolyl, phenoxy, indolyl,(1H-1,2,4-triazolyl)methyl and benzoxy; wherein R₆ and R₇ areindependently selected from hydrogen, C₁₋₄alkyl, C₃₋₈cycloalkyl,pyridinyl, phenyl, benzyl and naphthyl; wherein said phenyl, pyridinyl,benzyl, morpholino, morpholino-methyl, 1,3-dioxoisoindolinyl,1,2,4-oxadiazolyl, pyrazolyl, indolyl and benzoxy substituents of R₅ orsaid pyridinyl and phenyl of R₆ or R₇ can be unsubstituted or furthersubstituted with a group selected from halo, nitro, amino-sulfonyl,C₁₋₄alkyl, C₁₋₄alkoxy and halo-substituted-C₁₋₄alkyl; wherein X₃ isselected from a bond and C₁₋₄alkylene; or the pharmaceuticallyacceptable salt thereof; with the proviso that compounds of formula I donot include the compound where R₁ is hydrogen, R₂ is hydrogen and R₃ is—NHSO₂CH₂-phenyl; and the N-oxide derivatives, prodrug derivatives,protected derivatives, individual isomers and mixture of isomersthereof; and the pharmaceutically acceptable salts and solvates (e.g.hydrates) of such compounds.

In a second aspect, the present invention provides a pharmaceuticalcomposition which contains a compound of Formula I or a N-oxidederivative, individual isomers and mixture of isomers thereof; or apharmaceutically acceptable salt thereof, in admixture with one or moresuitable excipients.

In a third aspect, the present invention provides a method of treating adisease in an animal in which modulation of BCL-2 activity can prevent,inhibit or ameliorate the pathology and/or symptomology of the diseases,which method comprises administering to the animal a therapeuticallyeffective amount of a compound of Formula I or a N-oxide derivative,individual isomers and mixture of isomers thereof, or a pharmaceuticallyacceptable salt thereof.

In a fourth aspect, the present invention provides the use of a compoundof Formula I in the manufacture of a medicament for treating a diseasein an animal in which BCL-2 activity contributes to the pathology and/orsymptomology of the disease.

In a fifth aspect, the present invention provides a process forpreparing compounds of Formula I and the N-oxide derivatives, prodrugderivatives, protected derivatives, individual isomers and mixture ofisomers thereof, and the pharmaceutically acceptable salts thereof.

DEFINITIONS

The general terms used hereinbefore and hereinafter preferably havewithin the context of this disclosure the following meanings, unlessotherwise indicated, where more general terms wherever used may,independently of each other, be replaced by more specific definitions orremain, thus defining more detailed embodiments of the invention:

“Alkyl” as a group and as a structural element of other groups, forexample halo-substituted-alkyl and alkoxy, can be eitherstraight-chained or branched. C₁₋₄-alkoxy includes, methoxy, ethoxy, andthe like. Halo-substituted alkyl includes difluoromethyl,trifluoromethyl, pentafluoroethyl, and the like.

“Aryl” means a monocyclic or fused bicyclic aromatic ring assemblycontaining six to ten ring carbon atoms. For example, aryl may be phenylor naphthyl, preferably phenyl. “Arylene” means a divalent radicalderived from an aryl group.

“Heteroaryl” is as defined for aryl above where one or more of the ringmembers is a heteroatom. For example C₅₋₁₀heteroaryl is a minimum of 5members as indicated by the carbon atoms but that these carbon atoms canbe replaced by a heteroatom. Consequently, C₅₋₁₀heteroaryl includespyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, benzofuranyl,benzopyranyl, benzothiopyranyl, benzo[1,3]dioxole, imidazolyl,benzoimidazolyl, pyrimidinyl, furanyl, oxazolyl, isoxazolyl, triazolyl,tetrazolyl, pyrazolyl, thienyl, etc.

“Cycloalkyl” means a saturated or partially unsaturated, monocyclic,fused bicyclic or bridged polycyclic ring assembly containing the numberof ring atoms indicated. For example, C₃₋₁₀cycloalkyl includescyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

“Heterocycloalkyl” means cycloalkyl, as defined in this application,provided that one or more of the ring carbons indicated, are replaced bya moiety selected from —O—, —N═, —NR—, —C(O)—, —S—, —S(O)— or —S(O)₂—,wherein R is hydrogen, C₁₋₄alkyl or a nitrogen protecting group. Forexample, C₃₋₈heterocycloalkyl as used in this application to describecompounds of the invention includes morpholino, pyrrolidinyl,pyrrolidinyl-2-one, piperazinyl, piperidinyl, piperidinylone,1,4-dioxa-8-aza-spiro[4.5]dec-8-yl, thiomorpholino, sulfanomorpholino,sulfonomorpholino, etc.

“Halogen” (or halo) preferably represents chloro or fluoro, but may alsobe bromo or iodo.

Compounds of the formula I may have different isomeric forms. Forexample, any asymmetric carbon atom may be present in the (R)-, (S)- or(R,S)-configuration, preferably in the (R)- or (S)-configuration.Substituents at a double bond or especially a ring may be present incis-(═Z—) or trans (=E-) form. The compounds may thus be present asmixtures of isomers or preferably as pure isomers, preferably as purediastereomers or pure enantiomers.

Where the plural form (e.g. compounds, salts) is used, this includes thesingular (e.g. a single compound, a single salt). “A compound” does notexclude that (e.g. in a pharmaceutical formulation) more than onecompound of the formula I (or a salt thereof) is present, the “a” merelyrepresenting the indefinite article. “A” can thus preferably be read as“one or more”, less preferably alternatively as “one”.

Wherever a compound or compounds of the formula I are mentioned, this isfurther also intended to include N-oxides of such compounds and/ortautomers thereof.

The term “and/or an N-oxide thereof, a tautomer thereof and/or a(preferably pharmaceutically acceptable) salt thereof” especially meansthat a compound of the formula I may be present as such or in mixturewith its N-oxide, as tautomer (e.g. due to keto-enol, lactam-lactim,amide-imidic acid or enamine-imine tautomerism) or in (e.g. equivalencyreaction caused) mixture with its tautomer, or as a salt of the compoundof the formula I and/or any of these forms or mixtures of two or more ofsuch forms.

The present invention also includes all suitable isotopic variations ofthe compounds of the invention, or pharmaceutically acceptable saltsthereof. An isotopic variation of a compound of the invention or apharmaceutically acceptable salt thereof is defined as one in which atleast one atom is replaced by an atom having the same atomic number butan atomic mass different from the atomic mass usually found in nature.Examples of isotopes that may be incorporated into the compounds of theinvention and pharmaceutically acceptable salts thereof include, but arenot limited to, isotopes of hydrogen, carbon, nitrogen and oxygen suchas ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³⁵S, ¹⁸F, ³⁶Cl and ¹²³I.Certain isotopic variations of the compounds of the invention andpharmaceutically acceptable salts thereof, for example, those in which aradioactive isotope such as ³H or ¹⁴C is incorporated, are useful indrug and/or substrate tissue distribution studies. In particularexamples, ³H and ¹⁴C isotopes may be used for their ease of preparationand detectability. In other examples, substitution with isotopes such as²H may afford certain therapeutic advantages resulting from greatermetabolic stability, such as increased in vivo half-life or reduceddosage requirements. Isotopic variations of the compounds of theinvention or pharmaceutically acceptable salts thereof can generally beprepared by conventional procedures using appropriate isotopicvariations of suitable reagents.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to the discovery of compounds of Formula Icapable of inhibiting the interaction between BCL-2 and BH3. In oneembodiment, with respect to compounds of Formula I, are compounds ofFormula Ia:

in which:

R₁ is selected from hydrogen and halo;

R₂ is selected from hydrogen and C₁₋₄alkyl;

R₄ is selected from hydroxy and amino;

R₅ is selected from C₁₋₆alkyl, C₂₋₆alkenyl, cyclopropyl,imidazo[1,2-a]pyrimidinyl, 2-oxo-4-phenylpiperazin-1-yl,4-(2-chlorobenzyl)-3-oxopiperazin-1-yl, imidazo[1,2-a]pyridinyl,2-oxo-1,2,3,6-tetrahydropyrimidinyl,naphtho[2,1-d][1,2,3]oxadiazol-5-yl,(7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methyl,1H-pyrrolo[2,3-b]pyridinyl, 1H-pyrazolo[3,4-b]pyridinyl,benzo[c][1,2,5]thiadiazolyl, imidazo[2,1-b]thiazolyl,2,3-dihydrobenzo[b][1,4]dioxin-2-yl, naphtho[2,3-d][1,3]dioxol-2-yl,3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl, 1,2,4-oxadiazolyl,4-oxo-4,5,6,7-tetrahydrobenzofuranyl, 5-oxopyrrolidin-3-yl,benzo[d]isoxazolyl, 2,3-dihydrobenzofuran-3-yl, chroman-8-yl,3-oxo-3H-pyrazolyl, benzo[b]thiophenyl, benzo[b]furanyl,6-oxo-1,6-dihydropyridazinyl, 2-oxo-1,2-dihydropyridinyl,2-oxo-1,2,5,6,7,8-hexahydroquinolinyl,4-oxo-1,4-dihydro-1,8-naphthyridinyl, 4-oxo-4H-pyrano[2,3-b]pyridinyl,10,10-dioxido-9-oxo-9H-thioxanthen-3-yl, 1,2,5-oxadiazolyl,1,2,4-oxadiazolyl, dimethyl-amino, benzo[c][1,2,5]oxadiazolyl,isoindoline-1,3-dionyl, phenyl, benzyl, quinolinyl, isoquinolinyl,phenoxy, benzoxy, phenylthio, pyrrolyl, quinolin-8-yloxy,phenyl-sulfonyl, furanyl, thiazolyl, oxazolyl, isoxazolyl, thienyl,pyridinyl, pyrimidinyl, pyrrolidinyl, imidazolidine-2,4-dionyl,pyrrolidinonyl, piperidinyl, piperazinyl, pyrazinyl, pyrazolyl,morpholino, oxomorpholino, indolyl, benzo[d][1,2,3]triazol andoxopiperazinyl;

wherein said C₁₋₆alkyl, C₂₋₆alkenyl, cyclopropyl,imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyridinyl,1H-pyrrolo[2,3-b]pyridinyl, benzo[d]isoxazolyl,1H-pyrazolo[3,4-b]pyridinyl, 1,2,4-oxadiazolyl,benzo[c][1,2,5]thiadiazolyl, imidazo[2,1-b]thiazolyl,4-oxo-4,5,6,7-tetrahydrobenzofuranyl,2-oxo-1,2,3,6-tetrahydropyrimidinyl,2,3-dihydrobenzo[b][1,4]dioxin-2-yl, naphtho[2,3-d][1,3]dioxol-2-yl,3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl, 2,3-dihydrobenzofuran-3-yl,chroman-8-yl, 3-oxo-3H-pyrazolyl, 6-oxo-1,6-dihydropyridazinyl,2-oxo-1,2-dihydropyridinyl, benzo[b]thiophenyl, 1,2,5-oxadiazolyl,1,2,4-oxadiazolyl, benzyl, benzo[c][1,2,5]oxadiazolyl,isoindoline-1,3-dionyl, benzo[b]furanyl,2-oxo-1,2,5,6,7,8-hexahydroquinolinyl,4-oxo-1,4-dihydro-1,8-naphthyridinyl, 4-oxo-4H-pyrano[2,3-b]pyridinyl,10,10-dioxido-9-oxo-9H-thioxanthen-3-yl, 5-oxopyrrolidin-3-yl, phenyl,quinolinyl, pyrimidinyl, isoquinolinyl, phenoxy, benzoxy,phenoxy-methyl, phenylthio, pyrrolyl, quinolin-8-yloxy, phenyl-sulfonyl,furanyl, thiazolyl, oxazolyl, isoxazolyl, thienyl, pyridinyl,pyrrolidinyl, pyrrolidinonyl, piperazinyl, piperidinyl, pyrazinyl,pyrazolyl, morpholino, oxomorpholino, indolyl, benzo[d][1,2,3]triazol oroxopiperazinyl of R₅ is unsubstituted or substituted with 1 to 3 groupsindependently selected from halo, cyano, nitro, —NR₆R₇, C₁₋₄alkyl,halo-substituted-C₁₋₄alkyl, C₁₋₄alkoxy, halo-substituted-C₁₋₄alkoxy,halo-substituted-C₁₋₄alkylthio, —C(O)OR₆, —C(O)R₆, —X₃OR₆, —X₃NR₆C(O)R₇,—NR₆S(O)₂R₇, —C(O)NR₆R₇, —S(O)₀₋₂R₆, —S(O)₀₋₂NR₆R₇, phenyl, benzyl,morpholino, oxazolyl, naphthyl, pyrrolidinyl, piperidinyl,morpholino-methyl, 1,3-dioxoisoindolinyl, 1,2,4-oxadiazolyl, pyrazolyl,indolyl, (1H-1,2,4-triazolyl)methyl, phenoxy and benzoxy; wherein R₆ andR₇ are independently selected from hydrogen, C₁₋₄alkyl, C₃₋₈cycloalkyl,pyridinyl, pyrrolidinyl, phenyl, benzyl and naphthyl; wherein saidphenyl, pyridinyl, pyrrolidinyl, benzyl, morpholino, morpholino-methyl,1,2,4-oxadiazolyl, pyrazolyl, indolyl and benzoxy substituents of R₅ orsaid pyridinyl and phenyl of R₆ or R₇ can be unsubstituted or furthersubstituted with a group selected from halo, nitro, amino-sulfonyl,C₁₋₄alkyl, C₁₋₄alkoxy and halo-substituted-C₁₋₄alkyl; wherein X₃ isselected from a bond and C₁₋₄alkylene; or the pharmaceuticallyacceptable salt thereof.

In a further embodiment, R₁ and R₂ are hydrogen; R₄ is hydroxy; or thepharmaceutically acceptable salt thereof.

In a further embodiment are compounds selected from:

Pharmacology and Utility

The present invention makes available methods and compounds capable ofinhibiting the interaction between BCL-2 and proteins containing a BH3domain. One aspect of the present invention relates to a method oftreating a BCL-2-mediated disorder, comprising the step of administeringto a patient in need thereof a therapeutically effective amount of acompound of formula I as defined in the Summary of the Invention.

BCL-2 inhibitors have been shown to be active against a number of cancercell lines as a single agent, including, but not limited to, breastcancer (US 2003/0119894, published PCT applications WO 02/097053 and WO02/13833), lymphomas (Nature (2005) 435, 677-681), small cell lungcancer (Nature (2005) 435, 677-681), head and neck cancer (published PCTapplication WO 02/097053), and leukemias (published PCT application WO02/13833).

BCL-2 was originally identified at the chromosomal breakpoint of t(14;18)-bearing B-cell lymphomas and belongs to a growing family of proteinswhich regulate apoptosis. (Gross, A; McDonnell, J M; Korsmeyer, S. J.BCL-2 family members and the mitochondria in apoptosis. Genes &Development 1999, 13, 1899-1911, Cory, S.; Huang, D. C. S.; Adams, J. M.The BCL-2 family: roles in cell survival and oncogenesis. Oncogene, 200322, 8590-8607. Danial, N. N.; Korsmeyer, S. J. Cell death: Criticalcontrol points. Cell 2004, 116, 205-218. Chao, D. T.; Korsmeyer, S. J.BCL-2 family: regulators of cell death. Annu. Rev. Immunol. 1998, 16,395-419). Apoptosis, Christopher Potten, James Wilson, CambridgeUniversity Press, 2004). The BCL-2 family of proteins include bothanti-apoptotic molecules, such as BCL-2 and BCL-XL, and pro-apoptoticmolecules, such as BAX, BAK, BID and BAD. BCL-2 contributes to cancercell progression by preventing normal cell turnover caused byphysiological cell-death mechanisms. Over-expression of BCL-2 has beenobserved in 70% of breast cancer and many other forms of cancer(Buolaniwini, J. K. Novel anticancer drug discovery. Curr. Opin. Chem.Biol. 1999, 3, 500-509). The expression levels of BCL-2 proteins alsocorrelate with resistance to a wide spectrum of chemotherapeutic drugsand γ-radiation therapy (Reed, J. C.; Miyashita, T.; Takayama, S.; Wang,H.-G.; Sato, T.; Krajewski, S.; Aime-Sempe, C.; Bodrug, S.; Kitada, S.;Hanada, M. BCL-2 family proteins: Regulators of cell-death involved inthe pathogenesis of cancer and resistance to therapy. J. Cell. Biochem.1996, 60, 23-32; Reed, J. C. BCL-2 family proteins: strategies forovercoming chemoresistance in cancer. Advances in Pharmocology 1997, 41,501-553; Strasser, A.; Huang, D. C. S.; Vaux, D. L. The role of theBCL-2/ced-9 gene family in cancer and general implications of defects incell death control for tumorigenesis and resistance to chemotherapy.Biochem. Biophys. Acta 1997, 1333, F151-F189; DiPaola, R. S.; Aisner, J.Overcoming BCL-2- and p53-mediated resistance in prostate cancer. Semin.Oncol. 1999, 26, 112-116).

Members of the BCL-2 family of proteins represent key regulators ofapoptosis, with pro-apoptotic (e.g., BAX, BAK, BID, BIM, NOXA, PUMA) andanti-apoptotic function (e.g., BCL-2, BCL-XL, MCL-1). Selective andcompetitive dimerization between pro- and anti-apoptotic members of thefamily determines the fate of a cell given pro-apoptotic stimulus.Although the precise roles of BCL-2 and BCL-XL in cancer are notcompletely understood, there are several lines of evidence that suggestthat BCL-2 and BCL-XL not only contribute to cancer progression bypreventing normal cell turnover, but also play a role in the resistanceof cancer cells to current cancer treatments. Experimentalover-expression of BCL-2 (BCL-XL) renders cancer cells resistant to awide spectrum of chemotherapeutic agents and radiation (BCL-2 familyproteins: Regulators of cell-death involved in the pathogenesis ofcancer and resistance to therapy. J. Cell. Biochem. 1996, 60, 23-32;Reed, J. C). BCL-2 and/or BCL-XL are over-expressed in more than 50% ofall tumors as shown below (from Wang, S.; Yang, D.; Lippman, M. E.Targeting BCL-2 and BCL-XL with nonpeptidic small-molecule antagonists.Seminars in Oncology, 2003, 5, 133-142).

BCL-2 over- BCL-XL over- Cancer expression (%) expression (%) Prostate20-40 100  hormone resistant  80-100 — Breast 60-80 40-60 Non-small celllung 20-40 — Small cell lung 60-80 — Colorectal  50-100 83 Melanoma 6590 Multiple myeloma (at relapse) — 77 Head and Neck 13 52-75 Pancreatic23 90 Hepatocellular carcinoma — 80

Biological approaches to modulating BCL-2 function using anti-senseoligonucleotides or single-chain antibodies have been shown to enhancetumor cell chemosensitivity (Ziegler, A.; Luedke, G. H.; Fabbro, D.;Altmann, K. H.; Stahel, R. A.; Zangemeister-Wittke, U. Induction ofapoptosis in small-cell lung cancer cells by an antisenseoligodeoxynucleotide targeting the BCL-2 coding sequence. J. Natl.Cancer. Inst. 1997, 89, 1027-1036; Webb, A.; Cunningham, D.; Cotter, F.;Clarke, P. A.; Di Stefano, F.; Ross, P.; Corpo, M.; Dziewanowska, Z.BCL-2 antisense therapy in patients with non-hodgkin lymphoma. Lancet1997, 349, 1137-1141; Cotter, F. E. Phase I clinical and pharmacokineticstudy of BCL-2 antisense oligonucleotide therapy in patients withnon-hodgkin's lymphoma. J. Clin. Oncol. 2000, 18, 1812-1823; Piche, A.;Grim, J.; Rancourt, C.; Gomez-Navarro, J.; Reed, J. C.; Curiel, D. T.Modulation of BCL-2 protein levels by an intracellular anti-BCL-2single-chain antibody increases drug-induced cytotoxicity in the breastcancer cell line MCF-7. Cancer Res. 1998, 58, 2134-2140).

It has been shown that an anti-sense oligonucleotide (G3139) (Raynaud,F. I.; Orr, R. M.; Goddard, P. M.; Lacey, H. A.; Lancashire, H.; Judson,I. R.; Beck, T.; Bryan, B.; Cotter, F. E. Pharmacokinetics of G3139, aphosphorothioate oligodeoxynucleotide antisense to BCL-2, afterintravenous administration or continuous subcutaneous infusion to mice.J. Pharmacol. Exp. Ther. 1997, 281, 420-427), designed to hybridize tosequence in BCL-2 mRNA, inhibits BCL-2 expression, induces apoptosis andinhibits cell growth in human breast cancer cells having Bcl-2over-expression (Chen, H. X., Marchall, J. L., Trocky, N., Baidas, S.,Rizvi, N., Ling, Y., Bhagava, P., Lippman, M. E., Yang, D., and Hayes,D. F. A Phase I study of BCL-2 antisense G3139 (Genta) and weeklydocetaxel in patients with advanced breast cancer and other solidtumors. Proceedings of American Society of Clinical Oncology, 2000).Importantly, synergistic effects and complete tumor regression wereobserved in vivo in the combined treatments of G3139 with docetaxel.Therefore, BCL-2 represents a highly attractive target for thedevelopment of a novel therapy for the treatment of many forms ofcancers.

In certain embodiments, the present invention relates to theaforementioned method, wherein said BCL-2-mediated disorder is cancer.

In certain embodiments, the present invention relates to theaforementioned method, wherein said cancer is selected from the groupconsisting of acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic,monocytic, erythroleukemia, chronic leukemia, chronic myelocytic(granulocytic) leukemia, chronic lymphocytic leukemia, polycythemiaVera, Hodgkin's disease, non-Hodgkin's disease; multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,stadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterinecancer, testicular tumor, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, and endometrial cancer.

In certain embodiments, the present invention relates to theaforementioned method, wherein said cancer is follicular lymphoma,diffuse large B-cell lymphoma, mantle cell lymphoma, chronic lymphocyticleukemia prostrate cancer, breast cancer, neuroblastoma, colorectal,endometrial, ovarian, lung cancer, hepatocellular carcinoma, multiplemyeloma, head and neck or testicular cancer.

In certain embodiments, the present invention relates to theaforementioned method, wherein said cancer over-expresses BCL-2.

In certain embodiments, the present invention relates to theaforementioned method, wherein said cancer is dependent upon BCL-2 forgrowth and survival.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is administeredparenterally.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is administeredintramuscularly, intravenously, subcutaneously, orally, pulmonary,intrathecally, topically or intranasally.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is administeredsystemically.

In certain embodiments, the present invention relates to theaforementioned method, wherein said patient is a mammal.

In certain embodiments, the present invention relates to theaforementioned method, wherein said patient is a primate.

In certain embodiments, the present invention relates to theaforementioned method, wherein said patient is a human.

In another aspect, the present invention relates to a method of treatinga Bcl-mediated disorder, comprising the step of: administering to apatient in need thereof a therapeutically effective amount of achemotherapeutic agent in combination with a therapeutically effectiveamount of a compound of compound of formula I as defined in the Summaryof the Invention

Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the compounds described above, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,e.g., those targeted for buccal, sublingual, and systemic absorption,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation; (3) topical application,for example, as a cream, ointment, or a controlled-release patch orspray applied to the skin; (4) intravaginally or intrarectally, forexample, as a pessary, cream or foam; (5) sublingually; (6) ocularly;(7) transdermally; (8) nasally; (9) pulmonary; or (10) intrathecally.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect in at least a subpopulation of cells in an animal ata reasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately reacting a purifiedcompound of the invention in its free base form with a suitable organicor inorganic acid, and isolating the salt thus formed during subsequentpurification. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonatesalts and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cation,with ammonia, or with a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.(See, for example, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred per cent, this amount will range fromabout 0.1 per cent to about ninety-nine percent of active ingredient,preferably from about 5 per cent to about 70 per cent, most preferablyfrom about 10 percent to about 30 percent.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,celluloses, liposomes, micelle forming agents, e.g., bile acids, andpolymeric carriers, e.g., polyesters and polyanhydrides; and a compoundof the present invention. In certain embodiments, an aforementionedformulation renders orally bioavailable a compound of the presentinvention.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules, trouches and thelike), the active ingredient is mixed with one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds and surfactants, such as poloxamer and sodium laurylsulfate; (7) wetting agents, such as, for example, cetyl alcohol,glycerol monostearate, and non-ionic surfactants; (8) absorbents, suchas kaolin and bentonite clay; (9) lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, zinc stearate, sodium stearate, stearic acid, and mixturesthereof; (10) coloring agents; and (11) controlled release agents suchas crospovidone or ethyl cellulose. In the case of capsules, tablets andpills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-shelled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99% (morepreferably, 10 to 30%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the rate andextent of absorption, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, oral, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient, when used for the indicated analgesic effects,will range from about 0.0001 to about 100 mg per kilogram of body weightper day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. Preferred dosing is one administrationper day.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the subject compounds, as described above,formulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscularor intravenous injection as, for example, a sterile solution orsuspension; (3) topical application, for example, as a cream, ointmentor spray applied to the skin, lungs, or mucous membranes; or (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (5) sublingually or buccally; (6) ocularly; (7) transdermally; or(8) nasally.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable carriers and can also beadministered in conjunction with antimicrobial agents such aspenicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

Microemulsification technology can improve bioavailability of somelipophilic (water insoluble) pharmaceutical agents. Examples includeTrimetrine (Dordunoo, S. K., et al., Drug Development and IndustrialPharmacy, 17(12), 1685-1713, 1991 and REV 5901 (Sheen, P. C., et al., JPharm Sci 80(7), 712-714, 1991). Among other things, microemulsificationprovides enhanced bioavailability by preferentially directing absorptionto the lymphatic system instead of the circulatory system, which therebybypasses the liver, and prevents destruction of the compounds in thehepatobiliary circulation.

While all suitable amphiphilic carriers are contemplated, the presentlypreferred carriers are generally those that haveGenerally-Recognized-as-Safe (GRAS) status, and that can both solubilizethe compound of the present invention and microemulsify it at a laterstage when the solution comes into a contact with a complex water phase(such as one found in human gastro-intestinal tract). Usually,amphiphilic ingredients that satisfy these requirements have HLB(hydrophilic to lipophilic balance) values of 2-20, and their structurescontain straight chain aliphatic radicals in the range of C-6 to C-20.Examples are polyethylene-glycolized fatty glycerides and polyethyleneglycols.

Commercially available amphiphilic carriers are particularlycontemplated, including Gelucire-series, Labrafil, Labrasol, orLauroglycol (all manufactured and distributed by Gattefosse Corporation,Saint Priest, France), PEG-mono-oleate, PEG-dioleate, PEG-mono-laurateand di-laurate, Lecithin, Polysorbate 80, etc (produced and distributedby a number of companies in USA and worldwide).

Hydrophilic polymers suitable for use in the present invention are thosewhich are readily water-soluble, can be covalently attached to avesicle-forming lipid, and which are tolerated in vivo without toxiceffects (i.e., are biocompatible). Suitable polymers includepolyethylene glycol (PEG), polylactic (also termed polylactide),polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolicacid copolymer, and polyvinyl alcohol. Preferred polymers are thosehaving a molecular weight of from about 100 or 120 daltons up to about5,000 or 10,000 daltons, and more preferably from about 300 daltons toabout 5,000 daltons. In a particularly preferred embodiment, the polymeris polyethyleneglycol having a molecular weight of from about 100 toabout 5,000 daltons, and more preferably having a molecular weight offrom about 300 to about 5,000 daltons. In a particularly preferredembodiment, the polymer is polyethyleneglycol of 750 daltons (PEG(750)). Polymers may also be defined by the number of monomers therein;a preferred embodiment of the present invention utilizes polymers of atleast about three monomers, such PEG polymers consisting of threemonomers (approximately 150 daltons).

Other hydrophilic polymers which may be suitable for use in the presentinvention include polyvinylpyrrolidone, polymethoxazoline,polyethyloxazoline, polyhydroxypropyl methacrylamide,polymethacrylamide, polydimethylacrylamide, and derivatized cellulosessuch as hydroxymethylcellulose or hydroxyethylcellulose.

In certain embodiments, a formulation of the present invention comprisesa biocompatible polymer selected from the group consisting ofpolyamides, polycarbonates, polyalkylenes, polymers of acrylic andmethacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes,polyurethanes and co-polymers thereof, celluloses, polypropylene,polyethylenes, polystyrene, polymers of lactic acid and glycolic acid,polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid),poly(lactide-cocaprolactone), polysaccharides, proteins, polyhyaluronicacids, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

Cyclodextrins are cyclic oligosaccharides, consisting of 6, 7 or 8glucose units, designated by the Greek letter .alpha., .beta. or.gamma., respectively. Cyclodextrins with fewer than six glucose unitsare not known to exist. The glucose units are linked byalpha-1,4-glucosidic bonds. As a consequence of the chair conformationof the sugar units, all secondary hydroxyl groups (at C-2, C-3) arelocated on one side of the ring, while all the primary hydroxyl groupsat C-6 are situated on the other side. As a result, the external facesare hydrophilic, making the cyclodextrins water-soluble. In contrast,the cavities of the cyclodextrins are hydrophobic, since they are linedby the hydrogen of atoms C-3 and C-5, and by ether-like oxygens. Thesematrices allow complexation with a variety of relatively hydrophobiccompounds, including, for instance, steroid compounds such as17.beta.-estradiol (see, e.g., van Uden et al. Plant Cell Tiss. Org.Cult. 38:1-3-113 (1994)). The complexation takes place by Van der Waalsinteractions and by hydrogen bond formation. For a general review of thechemistry of cyclodextrins, see, Wenz, Agnew. Chem. Int. Ed. Engl.,33:803-822 (1994).

The physico-chemical properties of the cyclodextrin derivatives dependstrongly on the kind and the degree of substitution. For example, theirsolubility in water ranges from insoluble (e.g.,triacetyl-beta-cyclodextrin) to 147% soluble (w/v)(G-2-beta-cyclodextrin). In addition, they are soluble in many organicsolvents. The properties of the cyclodextrins enable the control oversolubility of various formulation components by increasing or decreasingtheir solubility.

Numerous cyclodextrins and methods for their preparation have beendescribed. For example, Parmeter (I), et al. (U.S. Pat. No. 3,453,259)and Gramera, et al. (U.S. Pat. No. 3,459,731) described electroneutralcyclodextrins. Other derivatives include cyclodextrins with cationicproperties [Parmeter (II), U.S. Pat. No. 3,453,257], insolublecrosslinked cyclodextrins (Solms, U.S. Pat. No. 3,420,788), andcyclodextrins with anionic properties [Parmeter (III), U.S. Pat. No.3,426,011]. Among the cyclodextrin derivatives with anionic properties,carboxylic acids, phosphorous acids, phosphinous acids, phosphonicacids, phosphoric acids, thiophosphonic acids, thiosulphinic acids, andsulfonic acids have been appended to the parent cyclodextrin [see,Parmeter (III), supra]. Furthermore, sulfoalkyl ether cyclodextrinderivatives have been described by Stella, et al. (U.S. Pat. No.5,134,127).

Liposomes consist of at least one lipid bilayer membrane enclosing anaqueous internal compartment. Liposomes may be characterized by membranetype and by size. Small unilamellar vesicles (SUVs) have a singlemembrane and typically range between 0.02 and 0.05 μm in diameter; largeunilamellar vesicles (LUVS) are typically larger than 0.05 μmOligolamellar large vesicles and multilamellar vesicles have multiple,usually concentric, membrane layers and are typically larger than 0.1μm. Liposomes with several nonconcentric membranes, i.e., severalsmaller vesicles contained within a larger vesicle, are termedmultivesicular vesicles.

One aspect of the present invention relates to formulations comprisingliposomes containing a compound of the present invention, where theliposome membrane is formulated to provide a liposome with increasedcarrying capacity. Alternatively or in addition, the compound of thepresent invention may be contained within, or adsorbed onto, theliposome bilayer of the liposome. The compound of the present inventionmay be aggregated with a lipid surfactant and carried within theliposome's internal space; in these cases, the liposome membrane isformulated to resist the disruptive effects of the activeagent-surfactant aggregate.

According to one embodiment of the present invention, the lipid bilayerof a liposome contains lipids derivatized with polyethylene glycol(PEG), such that the PEG chains extend from the inner surface of thelipid bilayer into the interior space encapsulated by the liposome, andextend from the exterior of the lipid bilayer into the surroundingenvironment.

Active agents contained within liposomes of the present invention are insolubilized form. Aggregates of surfactant and active agent (such asemulsions or micelles containing the active agent of interest) may beentrapped within the interior space of liposomes according to thepresent invention. A surfactant acts to disperse and solubilize theactive agent, and may be selected from any suitable aliphatic,cycloaliphatic or aromatic surfactant, including but not limited tobiocompatible lysophosphatidylcholines (LPCs) of varying chain lengths(for example, from about C.sub.14 to about C.sub.20).Polymer-derivatized lipids such as PEG-lipids may also be utilized formicelle formation as they will act to inhibit micelle/membrane fusion,and as the addition of a polymer to surfactant molecules decreases theCMC of the surfactant and aids in micelle formation. Preferred aresurfactants with CMCs in the micromolar range; higher CMC surfactantsmay be utilized to prepare micelles entrapped within liposomes of thepresent invention, however, micelle surfactant monomers could affectliposome bilayer stability and would be a factor in designing a liposomeof a desired stability.

Liposomes according to the present invention may be prepared by any of avariety of techniques that are known in the art. See, e.g., U.S. Pat.No. 4,235,871; Published PCT applications WO 96/14057; New RRC,Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104;Lasic DD, Liposomes from physics to applications, Elsevier SciencePublishers BV, Amsterdam, 1993.

For example, liposomes of the present invention may be prepared bydiffusing a lipid derivatized with a hydrophilic polymer into preformedliposomes, such as by exposing preformed liposomes to micelles composedof lipid-grafted polymers, at lipid concentrations corresponding to thefinal mole percent of derivatized lipid which is desired in theliposome. Liposomes containing a hydrophilic polymer can also be formedby homogenization, lipid-field hydration, or extrusion techniques, asare known in the art.

In one aspect of the present invention, the liposomes are prepared tohave substantially homogeneous sizes in a selected size range. Oneeffective sizing method involves extruding an aqueous suspension of theliposomes through a series of polycarbonate membranes having a selecteduniform pore size; the pore size of the membrane will correspond roughlywith the largest sizes of liposomes produced by extrusion through thatmembrane. See e.g., U.S. Pat. No. 4,737,323 (Apr. 12, 1988).

The release characteristics of a formulation of the present inventiondepend on the encapsulating material, the concentration of encapsulateddrug, and the presence of release modifiers. For example, release can bemanipulated to be pH dependent, for example, using a pH sensitivecoating that releases only at a low pH, as in the stomach, or a higherpH, as in the intestine. An enteric coating can be used to preventrelease from occurring until after passage through the stomach. Multiplecoatings or mixtures of cyanamide encapsulated in different materialscan be used to obtain an initial release in the stomach, followed bylater release in the intestine. Release can also be manipulated byinclusion of salts or pore forming agents, which can increase wateruptake or release of drug by diffusion from the capsule. Excipientswhich modify the solubility of the drug can also be used to control therelease rate. Agents which enhance degradation of the matrix or releasefrom the matrix can also be incorporated. They can be added to the drug,added as a separate phase (i.e., as particulates), or can beco-dissolved in the polymer phase depending on the compound. In allcases the amount should be between 0.1 and thirty percent (w/w polymer).Types of degradation enhancers include inorganic salts such as ammoniumsulfate and ammonium chloride, organic acids such as citric acid,benzoic acid, and ascorbic acid, inorganic bases such as sodiumcarbonate, potassium carbonate, calcium carbonate, zinc carbonate, andzinc hydroxide, and organic bases such as protamine sulfate, spermine,choline, ethanolamine, diethanolamine, and triethanolamine andsurfactants such as Tween® and Pluronic®. Pore forming agents which addmicrostructure to the matrices (i.e., water soluble compounds such asinorganic salts and sugars) are added as particulates. The range shouldbe between one and thirty percent (w/w polymer).

Uptake can also be manipulated by altering residence time of theparticles in the gut. This can be achieved, for example, by coating theparticle with, or selecting as the encapsulating material, a mucosaladhesive polymer. Examples include most polymers with free carboxylgroups, such as chitosan, celluloses, and especially polyacrylates (asused herein, polyacrylates refers to polymers including acrylate groupsand modified acrylate groups such as cyanoacrylates and methacrylates).

Pharmaceutical Combinations

The invention especially relates to the use of a compound of the formulaI (or a pharmaceutical composition comprising a compound of the formulaI) in the treatment of one or more of the diseases mentioned herein;wherein the response to treatment is beneficial as demonstrated, forexample, by the partial or complete removal of one or more of thesymptoms of the disease up to complete cure or remission.

Bcl-2 inhibitors have been shown to be active against a number of cancercell lines in combination with other anticancer agents and radiation,including, but not limited to, breast cancer (With docetaxel, publishedPCT application WO 02/097053), prostate cancer (With docetaxel,published PCT application WO 02/097053), head and neck cancer (Withdocetaxel, published PCT application WO 02/097053), and non small-celllung cancer (With paclitaxel, Nature (2005) 435, 677-681). In additionto the aforementioned combination chemotherapeutics, small moleculeinhibitors of Bcl-2 proteins display synergy with other anticanceragents, including, but not limited to etoposide, doxorubicin, cisplatin,paclitaxel, and radiation (Nature (2005) 435, 677-681).

A compound of formula (I) can also be used in combination with otherantiproliferative compounds. Such antiproliferative compounds include,but are not limited to aromatase inhibitors; antiestrogens;topoisomerase I inhibitors; topoisomerase II inhibitors; microtubuleactive compounds; alkylating compounds; histone deacetylase inhibitors;compounds which induce cell differentiation processes; cyclooxygenaseinhibitors; MMP inhibitors; mTOR inhibitors, such as RAD001;antineoplastic antimetabolites; platin compounds; compoundstargeting/decreasing a protein or lipid kinase activity and furtheranti-angiogenic compounds; compounds which target, decrease or inhibitthe activity of a protein or lipid phosphatase; gonadorelin agonists;anti-androgens; methionine aminopeptidase inhibitors; bisphosphonates;biological response modifiers; antiproliferative antibodies, such asHCD122; heparanase inhibitors; inhibitors of Ras oncogenic isoforms;telomerase inhibitors; proteasome inhibitors; compounds used in thetreatment of hematologic malignancies, such as FLUDARABINE; compoundswhich target, decrease or inhibit the activity of Flt-3, such as PKC412;Hsp90 inhibitors such as 17-AAG (17-allylaminogeldanamycin, NSC330507),17-DMAG (17-dimethylaminoethylamino-17-demethoxy-geldanamycin,NSC707545), IP1-504, CNF1010, CNF2024, CNF1010 from ConformaTherapeutics and AUY922; temozolomide (TEMODAL®); kinesin spindleprotein inhibitors, such as SB715992 or SB743921 from GlaxoSmithKline,or pentamidine/chlorpromazine from CombinatoRx; PI3K inhibitors, such asBEZ235; RAF inhibitors, such as LGX818 or RAF265; MEK inhibitors such asARRY142886 from Array PioPharma, AZD6244 from AstraZeneca, PD181461 fromPfizer, leucovorin, EDG binders, antileukemia compounds, ribonucleotidereductase inhibitors, S-adenosylmethionine decarboxylase inhibitors,antiproliferative antibodies or other chemotherapeutic compounds.Further, alternatively or in addition they may be used in combinationwith other tumor treatment approaches, including surgery, ionizingradiation, photodynamic therapy, implants, e.g. with corticosteroids,hormones, or they may be used as radiosensitizers. Also, inanti-inflammatory and/or antiproliferative treatment, combination withanti-inflammatory drugs is included. Combination is also possible withantihistamine drug substances, bronchodilatatory drugs, NSAID orantagonists of chemokine receptors.

The term “aromatase inhibitor” as used herein relates to a compoundwhich inhibits the estrogen production, i.e. the conversion of thesubstrates androstenedione and testosterone to estrone and estradiol,respectively. The term includes, but is not limited to steroids,especially atamestane, exemestane and formestane and, in particular,non-steroids, especially aminoglutethimide, roglethimide,pyridoglutethimide, trilostane, testolactone, ketokonazole, vorozole,fadrozole, anastrozole and letrozole. Exemestane can be administered,e.g., in the form as it is marketed, e.g. under the trademark AROMASIN.Formestane can be administered, e.g., in the form as it is marketed,e.g. under the trademark LENTARON. Fadrozole can be administered, e.g.,in the form as it is marketed, e.g. under the trademark AFEMA.Anastrozole can be administered, e.g., in the form as it is marketed,e.g. under the trademark ARIMIDEX. Letrozole can be administered, e.g.,in the form as it is marketed, e.g. under the trademark FEMARA or FEMAR.Aminoglutethimide can be administered, e.g., in the form as it ismarketed, e.g. under the trademark ORIMETEN. A combination of theinvention comprising a chemotherapeutic agent which is an aromataseinhibitor is particularly useful for the treatment of hormone receptorpositive tumors, e.g. breast tumors.

The term “antiestrogen” as used herein relates to a compound whichantagonizes the effect of estrogens at the estrogen receptor level. Theterm includes, but is not limited to tamoxifen, fulvestrant, raloxifeneand raloxifene hydrochloride. Tamoxifen can be administered, e.g., inthe form as it is marketed, e.g. under the trademark NOLVADEX.Raloxifene hydrochloride can be administered, e.g., in the form as it ismarketed, e.g. under the trademark EVISTA. Fulvestrant can be formulatedas disclosed in U.S. Pat. No. 4,659,516 or it can be administered, e.g.,in the form as it is marketed, e.g. under the trademark FASLODEX. Acombination of the invention comprising a chemotherapeutic agent whichis an antiestrogen is particularly useful for the treatment of estrogenreceptor positive tumors, e.g. breast tumors.

The term “anti-androgen” as used herein relates to any substance whichis capable of inhibiting the biological effects of androgenic hormonesand includes, but is not limited to, bicalutamide (CASODEX), which canbe formulated, e.g. as disclosed in U.S. Pat. No. 4,636,505.

The term “gonadorelin agonist” as used herein includes, but is notlimited to abarelix, goserelin and goserelin acetate. Goserelin isdisclosed in U.S. Pat. No. 4,100,274 and can be administered, e.g., inthe form as it is marketed, e.g. under the trademark ZOLADEX. Abarelixcan be formulated, e.g. as disclosed in U.S. Pat. No. 5,843,901.

The term “topoisomerase I inhibitor” as used herein includes, but is notlimited to topotecan, gimatecan, irinotecan, camptothecian and itsanalogues, 9-nitrocamptothecin and the macromolecular camptothecinconjugate PNU-166148 (compound A1 in WO99/17804). Irinotecan can beadministered, e.g. in the form as it is marketed, e.g. under thetrademark CAMPTOSAR. Topotecan can be administered, e.g., in the form asit is marketed, e.g. under the trademark HYCAMTIN.

The term “topoisomerase II inhibitor” as used herein includes, but isnot limited to the anthracyclines such as doxorubicin (includingliposomal formulation, e.g. CAELYX), daunorubicin, epirubicin,idarubicin and nemorubicin, the anthraquinones mitoxantrone andlosoxantrone, and the podophillotoxines etoposide and teniposide.Etoposide can be administered, e.g. in the form as it is marketed, e.g.under the trademark ETOPOPHOS. Teniposide can be administered, e.g. inthe form as it is marketed, e.g. under the trademark VM 26-BRISTOL.Doxorubicin can be administered, e.g. in the form as it is marketed,e.g. under the trademark ADRIBLASTIN or ADRIAMYCIN. Epirubicin can beadministered, e.g. in the form as it is marketed, e.g. under thetrademark FARMORUBICIN. Idarubicin can be administered, e.g. in the formas it is marketed, e.g. under the trademark ZAVEDOS. Mitoxantrone can beadministered, e.g. in the form as it is marketed, e.g. under thetrademark NOVANTRON.

The term “microtubule active compound” relates to microtubulestabilizing, microtubule destabilizing compounds and microtublinpolymerization inhibitors including, but not limited to taxanes, e.g.paclitaxel and docetaxel, vinca alkaloids, e.g., vinblastine, especiallyvinblastine sulfate, vincristine especially vincristine sulfate, andvinorelbine, discodermolides, cochicine and epothilones and derivativesthereof, e.g. epothilone B or D or derivatives thereof. Paclitaxel maybe administered e.g. in the form as it is marketed, e.g. TAXOL.Docetaxel can be administered, e.g., in the form as it is marketed, e.g.under the trademark TAXOTERE. Vinblastine sulfate can be administered,e.g., in the form as it is marketed, e.g. under the trademark VINBLASTINR.P. Vincristine sulfate can be administered, e.g., in the form as it ismarketed, e.g. under the trademark FARMISTIN. Discodermolide can beobtained, e.g., as disclosed in U.S. Pat. No. 5,010,099. Also includedare Epothilone derivatives which are disclosed in WO 98/10121, U.S. Pat.No. 6,194,181, WO 98/25929, WO 98/08849, WO 99/43653, WO 98/22461 and WO00/31247. Especially preferred are Epothilone A and/or B.

The term “alkylating compound” as used herein includes, but is notlimited to, cyclophosphamide, ifosfamide, melphalan or nitrosourea (BCNUor Gliadel). Cyclophosphamide can be administered, e.g., in the form asit is marketed, e.g. under the trademark CYCLOSTIN. Ifosfamide can beadministered, e.g., in the form as it is marketed, e.g. under thetrademark HOLOXAN.

The term “histone deacetylase inhibitors” or “HDAC inhibitors” relatesto compounds which inhibit the histone deacetylase and which possessantiproliferative activity. This includes compounds such as LDH589disclosed in WO 02/22577, especiallyN-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide,N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamideand pharmaceutically acceptable salts thereof. It further especiallyincludes Suberoylanilide hydroxamic acid (SAHA).

The term “antineoplastic antimetabolite” includes, but is not limitedto, 5-Fluorouracil or 5-FU, capecitabine, gemcitabine, DNA demethylatingcompounds, such as 5-azacytidine and decitabine, methotrexate andedatrexate, and folic acid antagonists such as pemetrexed. Capecitabinecan be administered, e.g., in the form as it is marketed, e.g. under thetrademark XELODA. Gemcitabine can be administered, e.g., in the form asit is marketed, e.g. under the trademark GEMZAR.

The term “platin compound” as used herein includes, but is not limitedto, carboplatin, cis-platin, cisplatinum and oxaliplatin. Carboplatincan be administered, e.g., in the form as it is marketed, e.g. under thetrademark CARBOPLAT. Oxaliplatin can be administered, e.g., in the formas it is marketed, e.g. under the trademark ELOXATIN.

The term “compounds targeting/decreasing a protein or lipid kinaseactivity”; or a “protein or lipid phosphatase activity”; or “furtheranti-angiogenic compounds” as used herein includes, but is not limitedto, protein tyrosine kinase and/or serine and/or threonine kinaseinhibitors or lipid kinase inhibitors, for example:

a) compounds targeting, decreasing or inhibiting the activity of theplatelet-derived growth factor-receptors (PDGFR), such as compoundswhich target, decrease or inhibit the activity of PDGFR, especiallycompounds which inhibit the PDGF receptor, e.g. aN-phenyl-2-pyrimidine-amine derivative, e.g. imatinib, SU101, SU6668 andGFB-111;

b) compounds targeting, decreasing or inhibiting the activity of thefibroblast growth factor-receptors (FGFR);

c) compounds targeting, decreasing or inhibiting the activity of theinsulin-like growth factor receptor I (IGF-IR), such as compounds whichtarget, decrease or inhibit the activity of IGF-IR, especially compoundswhich inhibit the kinase activity of IGF-I receptor, such as thosecompounds disclosed in WO 02/092599, or antibodies that target theextracellular domain of IGF-I receptor or its growth factors;

d) compounds targeting, decreasing or inhibiting the activity of the Trkreceptor tyrosine kinase family, or ephrin B4 inhibitors;

e) compounds targeting, decreasing or inhibiting the activity of the Axlreceptor tyrosine kinase family;

f) compounds targeting, decreasing or inhibiting the activity of the Retreceptor tyrosine kinase;

g) compounds targeting, decreasing or inhibiting the activity of theKit/SCFR receptor tyrosine kinase, i.e C-kit receptor tyrosinekinases—(part of the PDGFR family), such as compounds which target,decrease or inhibit the activity of the c-Kit receptor tyrosine kinasefamily, especially compounds which inhibit the c-Kit receptor, e.g.imatinib;

h) compounds targeting, decreasing or inhibiting the activity of membersof the c-Abl family, their gene-fusion products (e.g. BCR-Abl kinase)and mutants, such as compounds which target decrease or inhibit theactivity of c-AbI family members and their gene fusion products, e.g. aN-phenyl-2-pyrimidine-amine derivative, e.g. imatinib or nilotinib(AMN107); PD180970; AG957; NSC 680410; PD173955 from ParkeDavis; ordasatinib (BMS354825)

i) compounds targeting, decreasing or inhibiting the activity of membersof the protein kinase C (PKC) and Raf family of serine/threoninekinases, members of the MEK, SRC, JAK, FAK, PDK1, PKB/Akt, and Ras/MAPKfamily members, and/or members of the cyclindependent kinase family(CDK) and are especially those staurosporine derivatives disclosed inU.S. Pat. No. 5,093,330, e.g. midostaurin; examples of further compoundsinclude e.g. UCN-01, safingol, BAY 43-9006, Bryostatin 1, Perifosine;Ilmofosine; RO 318220 and RO 320432; GO 6976; Isis 3521;LY333531/LY379196; isochinoline compounds such as those disclosed in WO00/09495; FTIs; BEZ235 (a P13K inhibitor) or AT7519 (CDK inhibitor);

j) compounds targeting, decreasing or inhibiting the activity ofprotein-tyrosine kinase inhibitors, such as compounds which target,decrease or inhibit the activity of protein-tyrosine kinase inhibitorsinclude imatinib mesylate (GLEEVEC) or tyrphostin. A tyrphostin ispreferably a low molecular weight (Mr<1500) compound, or apharmaceutically acceptable salt thereof, especially a compound selectedfrom the benzylidenemalonitrile class or the S-arylbenzenemalonirile orbisubstrate quinoline class of compounds, more especially any compoundselected from the group consisting of Tyrphostin A23/RG-50810; AG 99;Tyrphostin AG 213; Tyrphostin AG 1748; Tyrphostin AG 490; TyrphostinB44; Tyrphostin B44 (+) enantiomer; Tyrphostin AG 555; AG 494;Tyrphostin AG 556, AG957 and adaphostin(4-{[(2,5-dihydroxyphenyl)methyl]amino}-benzoic acid adamantyl ester;NSC 680410, adaphostin);

k) compounds targeting, decreasing or inhibiting the activity of theepidermal growth factor family of receptor tyrosine kinases (EGFR,ErbB2, ErbB3, ErbB4 as homo- or heterodimers) and their mutants, such ascompounds which target, decrease or inhibit the activity of theepidermal growth factor receptor family are especially compounds,proteins or antibodies which inhibit members of the EGF receptortyrosine kinase family, e.g. EGF receptor, ErbB2, ErbB3 and ErbB4 orbind to EGF or EGF related ligands, and are in particular thosecompounds, proteins or monoclonal antibodies generically andspecifically disclosed in WO 97/02266, e.g. the compound of ex. 39, orin EP 0 564 409, WO 99/03854, EP 0520722, EP 0 566 226, EP 0 787 722, EP0 837 063, U.S. Pat. No. 5,747,498, WO 98/10767, WO 97/30034, WO97/49688, WO 97/38983 and, especially, WO 96/30347 (e.g. compound knownas CP 358774), WO 96/33980 (e.g. compound ZD 1839) and WO 95/03283 (e.g.compound ZM105180); e.g. trastuzumab (Herceptin™), cetuximab (Erbitux™),Iressa, Tarceva, OSI-774, CI-1033, EKB-569, GW-2016, E1.1, E2.4, E2.5,E6.2, E6.4, E2.11, E6.3 or E7.6.3, and 7H-pyrrolo-[2,3-d]pyrimidinederivatives which are disclosed in WO 03/013541; and

1) compounds targeting, decreasing or inhibiting the activity of thec-Met receptor, such as compounds which target, decrease or inhibit theactivity of c-Met, especially compounds which inhibit the kinaseactivity of c-Met receptor, or antibodies that target the extracellulardomain of c-Met or bind to HGF.

Further anti-angiogenic compounds include compounds having anothermechanism for their activity, e.g. unrelated to protein or lipid kinaseinhibition e.g. thalidomide (THALOMID) and TNP-470.

Compounds which target, decrease or inhibit the activity of a protein orlipid phosphatase are e.g. inhibitors of phosphatase 1, phosphatase 2A,or CDC25, e.g. okadaic acid or a derivative thereof.

Compounds which induce cell differentiation processes are e.g. retinoicacid, α- γ- or δ-tocopherol or α- γ- or δ-tocotrienol.

The term cyclooxygenase inhibitor as used herein includes, but is notlimited to, e.g. Cox-2 inhibitors, 5-alkyl substituted2-arylaminophenylacetic acid and derivatives, such as celecoxib(CELEBREX), rofecoxib (VIOXX), etoricoxib, valdecoxib or a5-alkyl-2-arylaminophenylacetic acid, e.g.5-methyl-2-(2′-chloro-6′-fluoroanilino)phenyl acetic acid, lumiracoxib.

The term “bisphosphonates” as used herein includes, but is not limitedto, etridonic, clodronic, tiludronic, pamidronic, alendronic,ibandronic, risedronic and zoledronic acid. “Etridonic acid” can beadministered, e.g., in the form as it is marketed, e.g. under thetrademark DIDRONEL. “Clodronic acid” can be administered, e.g., in theform as it is marketed, e.g. under the trademark BONEFOS. “Tiludronicacid” can be administered, e.g., in the form as it is marketed, e.g.under the trademark SKELID. “Pamidronic acid” can be administered, e.g.in the form as it is marketed, e.g. under the trademark AREDIA™.“Alendronic acid” can be administered, e.g., in the form as it ismarketed, e.g. under the trademark FOSAMAX. “Ibandronic acid” can beadministered, e.g., in the form as it is marketed, e.g. under thetrademark BONDRANAT. “Risedronic acid” can be administered, e.g., in theform as it is marketed, e.g. under the trademark ACTONEL. “Zoledronicacid” can be administered, e.g. in the form as it is marketed, e.g.under the trademark ZOMETA.

The term “mTOR inhibitors” relates to compounds which inhibit themammalian target of rapamycin (mTOR) and which possess antiproliferativeactivity such as sirolimus (Rapamune®), everolimus (Certican™), CCI-779and ABT578.

The term “heparanase inhibitor” as used herein refers to compounds whichtarget, decrease or inhibit heparin sulfate degradation. The termincludes, but is not limited to, PI-88.

The term “biological response modifier” as used herein refers to alymphokine or interferons, e.g. interferon γ.

The term “inhibitor of Ras oncogenic isoforms”, e.g. H-Ras, K-Ras, orN-Ras, as used herein refers to compounds which target, decrease orinhibit the oncogenic activity of Ras e.g. a “farnesyl transferaseinhibitor” e.g. L-744832, DK8G557 or R115777 (Zarnestra).

The term “telomerase inhibitor” as used herein refers to compounds whichtarget, decrease or inhibit the activity of telomerase. Compounds whichtarget, decrease or inhibit the activity of telomerase are especiallycompounds which inhibit the telomerase receptor, e.g. telomestatin.

The term “methionine aminopeptidase inhibitor” as used herein refers tocompounds which target, decrease or inhibit the activity of methionineaminopeptidase. Compounds which target, decrease or inhibit the activityof methionine aminopeptidase are e.g. bengamide or a derivative thereof.

The term “proteasome inhibitor” as used herein refers to compounds whichtarget, decrease or inhibit the activity of the proteasome. Compoundswhich target, decrease or inhibit the activity of the proteasome includee.g. Bortezomid (Velcade™) and MLN 341.

The term “matrix metalloproteinase inhibitor” or (“MMP” inhibitor) asused herein includes, but is not limited to, collagen peptidomimetic andnonpeptidomimetic inhibitors, tetracycline derivatives, e.g. hydroxamatepeptidomimetic inhibitor batimastat and its orally bioavailable analoguemarimastat (BB-2516), prinomastat (AG3340), metastat (NSC 683551)BMS-279251, BAY 12-9566, TAA211, MMI270B or AAJ996.

The term “compounds used in the treatment of hematologic malignancies”as used herein includes, but is not limited to, FMS-like tyrosine kinaseinhibitors e.g. compounds targeting, decreasing or inhibiting theactivity of FMS-like tyrosine kinase receptors (Flt-3R); interferon,1-b-D-arabinofuransylcytosine (ara-c) and bisulfan; and ALK inhibitorse.g. compounds which target, decrease or inhibit anaplastic lymphomakinase.

Compounds which target, decrease or inhibit the activity of FMS-liketyrosine kinase receptors (Flt-3R) are especially compounds, proteins orantibodies which inhibit members of the Flt-3R receptor kinase family,e.g. PKC412, TKI258, midostaurin, a staurosporine derivative, SU11248and MLN518.

The term “HSP90 inhibitors” as used herein includes, but is not limitedto, compounds targeting, decreasing or inhibiting the intrinsic ATPaseactivity of HSP90; degrading, targeting, decreasing or inhibiting theHSP90 client proteins via the ubiquitin proteosome pathway. Compoundstargeting, decreasing or inhibiting the intrinsic ATPase activity ofHSP90 are especially compounds, proteins or antibodies which inhibit theATPase activity of HSP90 e.g., 17-allylamino, 17-demethoxygeldanamycin(17AAG), a geldanamycin derivative; other geldanamycin relatedcompounds; radicicol and HDAC inhibitors. An example HSP90 inhibitor isAUY922.

The term “antiproliferative antibodies” as used herein includes, but isnot limited to, trastuzumab (Herceptin™), Trastuzumab-DM1, erbitux,bevacizumab (Avastin™) rituximab (Rituxan®), PRO64553 (anti-CD40), 2C4Antibody and HCD122 antibody (anti-CD40). By antibodies is meant e.g.intact monoclonal antibodies, polyclonal antibodies, multispecificantibodies formed from at least 2 intact antibodies, and antibodiesfragments so long as they exhibit the desired biological activity.

For the treatment of acute myeloid leukemia (AML), compounds of formula(I) can be used in combination with standard leukemia therapies,especially in combination with therapies used for the treatment of AML.In particular, compounds of formula (I) can be administered incombination with, e.g., farnesyl transferase inhibitors and/or otherdrugs useful for the treatment of AML, such as Daunorubicin, Adriamycin,Ara-C, VP-16, Teniposide, Mitoxantrone, Idarubicin, Carboplatinum andPKC412.

The term “antileukemic compounds” includes, for example, Ara-C, apyrimidine analog, which is the 2′-alpha-hydroxy ribose (arabinoside)derivative of deoxycytidine. Also included is the purine analog ofhypoxanthine, 6-mercaptopurine (6-MP) and fludarabine phosphate.

Compounds which target, decrease or inhibit activity of histonedeacetylase (HDAC) inhibitors such as sodium butyrate andsuberoylanilide hydroxamic acid (SAHA) inhibit the activity of theenzymes known as histone deacetylases. Specific HDAC inhibitors includeMS275, SAHA, FK228 (formerly FR901228), Trichostatin A and compoundsdisclosed in U.S. Pat. No. 6,552,065, in particular,N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]-methyl]phenyl]-2E-2-propenamide,or a pharmaceutically acceptable salt thereof andN-hydroxy-3-[4-[(2-hydroxyethyl){2-(1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide,or a pharmaceutically acceptable salt thereof, especially the lactatesalt.

Somatostatin receptor antagonists as used herein refers to compoundswhich target, treat or inhibit the somatostatin receptor such asoctreotide, and SOM230 (pasireotide).

Tumor cell damaging approaches refer to approaches such as ionizingradiation. The term “ionizing radiation” referred to above andhereinafter means ionizing radiation that occurs as eitherelectromagnetic rays (such as X-rays and gamma rays) or particles (suchas alpha and beta particles). Ionizing radiation is provided in, but notlimited to, radiation therapy and is known in the art. See Hellman,Principles of Radiation Therapy, Cancer, in Principles and Practice ofOncology, Devita et al., Eds., 4^(th) Edition, Vol. 1, pp. 248-275(1993).

The term “EDG binders” as used herein refers a class ofimmunosuppressants that modulates lymphocyte recirculation, such asFTY720.

The term “ribonucleotide reductase inhibitors” refers to pyrimidine orpurine nucleoside analogs including, but not limited to, fludarabineand/or cytosine arabinoside (ara-C), 6-thioguanine, 5-fluorouracil,cladribine, 6-mercaptopurine (especially in combination with ara-Cagainst ALL) and/or pentostatin. Ribonucleotide reductase inhibitors areespecially hydroxyurea or 2-hydroxy-1H-isoindole-1,3-dione derivatives,such as PL-1, PL-2, PL-3, PL-4, PL-5, PL-6, PL-7 or PL-8 mentioned inNandy et al., Acta Oncologica, Vol. 33, No. 8, pp. 953-961 (1994).

The term “S-adenosylmethionine decarboxylase inhibitors” as used hereinincludes, but is not limited to the compounds disclosed in U.S. Pat. No.5,461,076.

Also included are in particular those compounds, proteins or monoclonalantibodies of VEGF disclosed in WO 98/35958, e.g.1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or a pharmaceuticallyacceptable salt thereof, e.g. the succinate, or in WO 00/09495, WO00/27820, WO 00/59509, WO 98/11223, WO 00/27819 and EP 0 769 947; thoseas described by Prewett et al, Cancer Res, Vol. 59, pp. 5209-5218(1999); Yuan et al., Proc Natl Acad Sci USA, Vol. 93, pp. 14765-14770(1996); Zhu et al., Cancer Res, Vol. 58, pp. 3209-3214 (1998); andMordenti et al., Toxicol Pathol, Vol. 27, No. 1, pp. 14-21 (1999); in WO00/37502 and WO 94/10202; ANGIOSTATIN, described by O'Reilly et al.,Cell, Vol. 79, pp. 315-328 (1994); ENDOSTATIN, described by O'Reilly etal., Cell, Vol. 88, pp. 277-285 (1997); anthranilic acid amides; ZD4190;ZD6474; SU5416; SU6668; bevacizumab; or anti-VEGF antibodies oranti-VEGF receptor antibodies, e.g. rhuMAb and RHUFab, VEGF aptamer e.g.Macugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgG1 antibody,Angiozyme (RPI 4610) and Bevacizumab (Avastin™).

Photodynamic therapy as used herein refers to therapy which uses certainchemicals known as photosensitizing compounds to treat or preventcancers. Examples of photodynamic therapy includes treatment withcompounds, such as e.g. VISUDYNE and porfimer sodium.

Angiostatic steroids as used herein refers to compounds which block orinhibit angiogenesis, such as, e.g., anecortave, triamcinolone.hydrocortisone, 11-α-epihydrocotisol, cortexolone,17α-hydroxyprogesterone, corticosterone, desoxycorticosterone,testosterone, estrone and dexamethasone.

Implants containing corticosteroids refers to compounds, such as e.g.fluocinolone, dexamethasone.

“Other chemotherapeutic compounds” include, but are not limited to,plant alkaloids, hormonal compounds and antagonists; biological responsemodifiers, preferably lymphokines or interferons; antisenseoligonucleotides or oligonucleotide derivatives; shRNA or siRNA; ormiscellaneous compounds or compounds with other or unknown mechanism ofaction.

The structure of the active compounds identified by code nos., genericor trade names may be taken from the actual edition of the standardcompendium “The Merck Index” or from databases, e.g. PatentsInternational (e.g. IMS World Publications).

None of the quotations of references made within the present disclosureis to be understood as an admission that the references cited are priorart that would negatively affect the patentability of the presentinvention.

Processes for Making Compounds of the Invention

The present invention also includes processes for the preparation ofcompounds of the invention. In the reactions described, it can benecessary to protect reactive functional groups, for example hydroxy,amino, imino, thio or carboxy groups, where these are desired in thefinal product, to avoid their unwanted participation in the reactions.Conventional protecting groups can be used in accordance with standardpractice, for example, see T. W. Greene and P. G. M. Wuts in “ProtectiveGroups in Organic Chemistry”, John Wiley and Sons, 1991.

Compounds of Formula I, in which R₃ is linked to the phenyl ring via asulfonamide, can be prepared by proceeding as in the following ReactionScheme IV:

in which R₁, R₂, R₄ and R₅ are as defined for Formula I in the Summaryof the Invention. A compound of Formula I can be prepared by reacting acompound of (4) with a compound of formula (5) a suitable base (such astriethylamine, and the like), and a suitable solvent (such as DCM, andthe like). The reaction takes place at about 120° C. and can take up toabout 2 hours to complete. For reaction scheme IV, the positions of R₂and NH₂ on the phenyl ring of compound 2 are interchangeable.

Detailed examples of the synthesis of compounds of Formula I can befound in the Examples, infra.

Additional Processes for Making Compounds of the Invention

A compound of the invention can be prepared as a pharmaceuticallyacceptable acid addition salt by reacting the free base form of thecompound with a pharmaceutically acceptable inorganic or organic acid.Alternatively, a pharmaceutically acceptable base addition salt of acompound of the invention can be prepared by reacting the free acid formof the compound with a pharmaceutically acceptable inorganic or organicbase.

Compounds of the formula I can also be modified by appending appropriatefunctionalities to enhance selective biological properties.Modifications of this kind are known in the art and include those thatincrease penetration into a given biological system (e.g. blood,lymphatic system, central nervous system, testis), increasebioavailability, increase solubility to allow parenteral administration(e.g. injection, infusion), alter metabolism and/or alter the rate ofsecretion. Examples of this type of modifications include but are notlimited to esterification, e.g. with polyethylene glycols,derivatisation with pivaloyloxy or fatty acid substituents, conversionto carbamates, hydroxylation of aromatic rings and heteroatomsubstitution in aromatic rings. Wherever compounds of the formula I,and/or N-oxides, tautomers and/or (preferably pharmaceuticallyacceptable) salts thereof are mentioned, this comprises such modifiedformulae, while preferably the molecules of the formula I, theirN-oxides, their tautomers and/or their salts are meant.

Alternatively, the salt forms of the compounds of the invention can beprepared using salts of the starting materials or intermediates. In viewof the close relationship between the novel compounds of the formula Iin free form and those in the form of their salts, including those saltsthat can be used as intermediates, for example in the purification oridentification of the novel compounds, any reference to the compounds ora compound of the formula I hereinbefore and hereinafter is to beunderstood as referring to the compound in free form and/or also to oneor more salts thereof, as appropriate and expedient, as well as to oneor more solvates, e.g. hydrates.

Salts are formed, for example, as acid addition salts, preferably withorganic or inorganic acids, from compounds of formula I with a basicnitrogen atom, especially the pharmaceutically acceptable salts.Suitable inorganic acids are, for example, halogen acids, such ashydrochloric acid, sulfuric acid, or phosphoric acid. Suitable organicacids are, for example, carboxylic, phosphonic, sulfonic or sulfamicacids, for example acetic acid, propionic acid, octanoic acid, decanoicacid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid,succinic acid, malonic acid, adipic acid, pimelic acid, suberic acid,azelaic acid, malic acid, tartaric acid, citric acid, amino acids, suchas glutamic acid or aspartic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, cyclohexanecarboxylic acid, adamantanecarboxylicacid, benzoic acid, salicylic acid, 4-aminosalicylic acid, phthalicacid, phenylacetic acid, mandelic acid, cinnamic acid, methane- orethane-sulfonic acid, 2-hydroxyethanesulfonic acid,ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-toluenesulfonicacid, 2-naphthalenesulfonic acid, 1,5-naphthalenedisulfonic acid, 2- or3-methylbenzenesulfonic acid, methylsulfuric acid, ethylsulfuric acid,dodecylsulfuric acid, N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- orN-propyl-sulfamic acid, or other organic protonic acids, such asascorbic acid.

For isolation or purification purposes it is also possible to usepharmaceutically unacceptable salts, for example picrates orperchlorates. For therapeutic use, only pharmaceutically acceptablesalts or free compounds are employed (where applicable in the form ofpharmaceutical preparations), and these are therefore preferred.

The free acid or free base forms of the compounds of the invention canbe prepared from the corresponding base addition salt or acid additionsalt from, respectively. For example a compound of the invention in anacid addition salt form can be converted to the corresponding free baseby treating with a suitable base (e.g., ammonium hydroxide solution,sodium hydroxide, and the like). A compound of the invention in a baseaddition salt form can be converted to the corresponding free acid bytreating with a suitable acid (e.g., hydrochloric acid, etc.).

Compounds of the invention in unoxidized form can be prepared fromN-oxides of compounds of the invention by treating with a reducing agent(e.g., sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride,sodium borohydride, phosphorus trichloride, tribromide, or the like) ina suitable inert organic solvent (e.g. acetonitrile, ethanol, aqueousdioxane, or the like) at 0 to 80° C.

Prodrug derivatives of the compounds of the invention can be prepared bymethods known to those of ordinary skill in the art (e.g., for furtherdetails see Saulnier et al., (1994), Bioorganic and Medicinal ChemistryLetters, Vol. 4, p. 1985). For example, appropriate prodrugs can beprepared by reacting a non-derivatized compound of the invention with asuitable carbamylating agent (e.g., 1,1-acyloxyalkylcarbanochloridate,paranitrophenyl carbonate, or the like).

Protected derivatives of the compounds of the invention can be made bymeans known to those of ordinary skill in the art. A detaileddescription of techniques applicable to the creation of protectinggroups and their removal can be found in T. W. Greene, “ProtectingGroups in Organic Chemistry”, 3^(rd) edition, John Wiley and Sons, Inc.,1999.

Compounds of the present invention can be conveniently prepared, orformed during the process of the invention, as solvates (e.g.,hydrates). Hydrates of compounds of the present invention can beconveniently prepared by recrystallization from an aqueous/organicsolvent mixture, using organic solvents such as dioxin, tetrahydrofuranor methanol.

Compounds of the invention can be prepared as their individualstereoisomers by reacting a racemic mixture of the compound with anoptically active resolving agent to form a pair of diastereoisomericcompounds, separating the diastereomers and recovering the opticallypure enantiomers. While resolution of enantiomers can be carried outusing covalent diastereomeric derivatives of the compounds of theinvention, dissociable complexes are preferred (e.g., crystallinediastereomeric salts). Diastereomers have distinct physical properties(e.g., melting points, boiling points, solubilities, reactivity, etc.)and can be readily separated by taking advantage of thesedissimilarities. The diastereomers can be separated by chromatography,or preferably, by separation/resolution techniques based upondifferences in solubility. The optically pure enantiomer is thenrecovered, along with the resolving agent, by any practical means thatwould not result in racemization. A more detailed description of thetechniques applicable to the resolution of stereoisomers of compoundsfrom their racemic mixture can be found in Jean Jacques, Andre Collet,Samuel H. Wilen, “Enantiomers, Racemates and Resolutions”, John WileyAnd Sons, Inc., 1981.

In summary, the compounds of Formula I can be made by a process, whichinvolves:

(a) those of reaction schemes I; and

(b) optionally converting a compound of the invention into apharmaceutically acceptable salt;

(c) optionally converting a salt form of a compound of the invention toa non-salt form;

(d) optionally converting an unoxidized form of a compound of theinvention into a pharmaceutically acceptable N-oxide;

(e) optionally converting an N-oxide form of a compound of the inventionto its unoxidized form;

(f) optionally resolving an individual isomer of a compound of theinvention from a mixture of isomers;

(g) optionally converting a non-derivatized compound of the inventioninto a pharmaceutically acceptable prodrug derivative; and

(h) optionally converting a prodrug derivative of a compound of theinvention to its non-derivatized form.

Insofar as the production of the starting materials is not particularlydescribed, the compounds are known or can be prepared analogously tomethods known in the art or as disclosed in the Examples hereinafter.

One of skill in the art will appreciate that the above transformationsare only representative of methods for preparation of the compounds ofthe present invention, and that other well known methods can similarlybe used.

EXAMPLES

The following intermediates and examples serve to illustrate theinvention without limiting the scope thereof. The followingabbreviations and methods are used in the descriptions of the examples:

ABBREVIATIONS

aq. (aqueous); AcOH (acetic acid); DCM (dichloromethane); DIPEA(diisopropylethylamine); DME (1,2-dimethoxyethane); DMSO(dimethylsulfoxide); EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride); eq (equivalent(s)); Et₃N (triethylamine); EtOAc (ethylacetate); EtOH (ethanol); h (hour(s)); HOBt (1-hydroxybenzotriazole);MeOH (methanol); min (minute(s)); MS (mass spectrometry); N (normality);NMR (nuclear magnetic resonance spectrometry); Rf (retention factor); RT(room temperature); TBDMS (tertiary butyl dimethyl silyl); THF(tetrahydrofuran); and TLC (thin layer chromatography).

HPLC Conditions:

Method A: Column: Inertsil ODS3V (250×4.6) mm, 5 μm; Mobile phase: A:0.01M KH₂PO₄/0.01M KH₂PO₄ pH adjusted to 6.5; B: ACN; GradientInformation: (T/% B): 0/30, 2/30, 6/85, 16/85, 17/30, 18/30); Flow Rate:1.0 ml/min; Detection by UV at: 210.0 nm.

Method B: Column: Inertsil ODS3V (250×4.6) mm, 5 μm; Mobile phase: A:0.01M KH₂PO₄/0.01M KH₂PO₄ pH adjusted to 6.5; B: ACN; GradientInformation: (T/% B): 0/30, 2/30, 6/80, 13/80, 14/30, 15/30); Flow Rate:1.0 ml/min; Detection by UV at: 210.0 nm.

Method C: Column: XTerra RP18 (250×4.6) mm, 5 μm; Mobile phase: A: 0.01MKH₂PO₄/0.01M KH₂PO₄ pH adjusted to 6.5; B: ACN; Gradient Information:(T/% B): 0/30, 2/30, 6/85, 16/85, 17/30, 18/30); Flow Rate: 1.0 ml/min;Detection by UV at: 210.0 nm.

Method D: Column: XTerra RP18 (250×4.6) mm, 5 μm; Mobile phase: A: 0.01MKH₂PO₄/0.01M KH₂PO₄ pH adjusted to 6.5; B: ACN; Gradient Information:(T/% B): 0/30, 2/30, 6/80, 13/80, 14/30, 15/30); Flow Rate: 1.0 ml/min;Detection by UV at: 210.0 nm.

Method E: Column: Hypersil BDS C18 (250×4.6) mm, 5 μm; Mobile phase: A:0.01M KH₂PO₄/0.01M KH₂PO₄ pH adjusted to 6.5; B: ACN; GradientInformation: (T/% B): 0/30, 15/50, 18/90, 28/90, 28.10/30); Flow Rate:0.8 ml/min; Detection by UV at: 260.0 nm.

Method F: Column: Hypersil BDS C18 (250×4.6) mm, 5 μm; Mobile phase: A:0.01M ammonium acetate; B: ACN; Gradient Information: (T/% B): 0/30,15/50, 18/90, 28/90, 28.10/30); Flow Rate: 0.8 ml/min; Detection by UVat: 260.0 nm.

Method G: Column: XTerra RP18 (250×4.0) mm, 5 μm; Mobile phase: A: 0.01MKH2PO4 (pH 6.5); B: ACN; Gradient Information: (T/% B): 0/30, 2/30,6/80, 13/80, 14/30, 15/30; Flow Rate: 1.0 ml/min; Detection by UV at:210.0 nm.

Method H: Column: Inertsil ODS3V (250×4.6) mm, 5 μm; Mobile phase: A:0.01M KH2PO4 (pH adjusted to 6.5); B: ACN; Gradient Information: (T/%B): 0/70, 1.5/70, 5/85, 13/85, 14/70, 15/70; Flow Rate: 1.0 ml/min;Detection by UV at: 210.0 nm.

Method I: Column: XTerra RP18 (250×4.6) mm, 5 μm; Mobile phase: A: 0.01MKH2PO4; B: ACN; Gradient Information: (T/% B): 0/30, 2/30, 6/80, 16/80,17/30, 18/30; Flow Rate: 1.0 ml/min; Detection by UV at: 210.0 nm.

Method J: Column: ACES C18 (250×4.6) mm, 5 μm; Mobile phase: A: 0.01MKH2PO4; B: ACN; Gradient Information: (T/% B): 0/30, 2/30, 6/85, 16/85,17/30, 18/30; Flow Rate: 1.0 ml/min; Detection by UV at: 210.0 nm.

Method K: Column: Inertsil ODS3V; Mobile phase: A: 0.01M KH2PO4; B: ACN;Gradient Information: (T/% B): 0/50, 1.5/50, 5/80, 13/80, 14/50, 15/50;Flow Rate: 1.0 ml/min; Detection by UV at: 210.0 nm.

Method L: Column: XTerra RP18 (250×4.6) mm, 5 μm; Mobile phase: A: 0.01MKH2PO4 (pH 6.5); B: ACN; Gradient Information: (T/% B): 0/50, 2/50,9/85, 16/85, 17/50, 18/50; Flow Rate: 1.0 ml/min; Detection by UV at:210.0 nm.

Method M: Column: Symmetry Shield RP18 (150 mm×4.6 mm), 5 μm; Mobilephase: A: 0.01% TFA (aq.); B: ACN; Gradient Information: (T/% B): 0/20,2/20, 6/85, 13/85, 14/20, 15/20; Flow Rate: 1.0 ml/min; Detection by UVat: 210.0 nm.

NMR Spectra:

1H NMR spectra were recorded on a Varian 400 MHz (Varian Mercury Plus)or 500 MHz (Unity NOVA) spectrometers with DMSO-d₆ or CDCl3 as thesolvents. Chemical shifts were reported in 6 scale usingtetramethylsilane (TMS, d 0.00) as internal standard and couplingconstants (J) were reported in Hz. The standard abbreviations s, d, t,q, dd, dt and m were used to symbolize singlet, doublet, triplet,quartet, double doublet, doublet of a triplet and multipletrespectively.

Mass Spectra:

The LC-MS and ES-MS spectra were performed on Perkin-Elmer Sciex, modelAPI 3000.

LC-MS Conditions:

Method A: Regular method in Formic Acid (FA); Column: Cynergi 2.5 μmMax-RP100A (20×4.0) mm; Mobile Phase: A: 0.1% FA (aq.); B: ACN; T/% B:0/20, 0.5/20, 2.5/95, 4.5/95, 5.0/20; Flow: 1.5 mL/min.

Method B: Regular method in Ammonium Acetate (AA); Column: Cynergi 2.5μm Max-RP100A (20×4.0) mm; Mobile Phase: A: 0.01M Ammonium acetate(aq.); B: ACN; T % B: 0/20, 1.0/20, 2.5/85, 4.0/95, 4.5/20, 5.0/20;Flow: 1.0 mL/min.

Intermediate A(S)-1-(4-amino-2-(3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)phenyl)-N,N-dibutyl-5-methyl-1H-pyrazole-3-carboxamide

Step 1: Preparation of 2-hydrazinyl-5-nitrobenzoic acid hydrochloride

To an ice cooled solution of 2-amino-5-nitrobenzoic acid (50.0 g, 274.5mmol) in water (350 mL) was added conc. HCl (404 mL). The reactionmixture was stirred until the salt had precipitated out. A solution ofsodium nitrite (29.0 g, 411.8 mmol) in water (300 mL) was slowly addedat 0° C. with stirring for 15 min. This reaction mixture was slowlyadded to ice-cold sulfurous acid (2.5 L) followed by stirring at RT for12 h. The reaction mixture was again cooled to 0° C. and conc. HCl wasadded until solid separates out. The solid was filtered, washed withcold HCl and vacuum dried to afford the title compound as a yellowcolored solid 60 g (93%), which was used without further purification.Rf=0.10 (10% MeOH in DCM).

Step 2: Preparation of2-(3-(ethoxycarbonyl)-5-methyl-1H-pyrazol-1-yl)-5-nitrobenzoic acid

To a solution of 2-hydrazinyl-5-nitrobenzoic acid hydrochloride (59.4 g,255.1 mmol) in AcOH (500 mL) was added ethyl-2,4-dioxovalerate (31.0 g,196.2 mmol). The reaction mixture was refluxed for 2 h. AcOH wasevaporated invacuo and the residue was co-distilled with toluene (100mL) twice. The residue was dissolved in EtOAc and washed with wateruntil the aq. layer became neutral. The organic layer was dried oversodium sulphate and concentrated invacuo. The gummy material wastriturated with diethyl ether to afford the title compound as a whitesolid 35 g (56%). Rf=0.30 (10% MeOH in DCM); ¹H NMR (400 MHz, DMSO-d₆):δ 13.90-13.60 (brs, 1H), 8.62 (d, J=2.5 Hz, 1H), 8.54 (dd, J=2.6 & 8.5Hz, 1H), 7.90 (d, J=8.3 Hz, 1H), 6.76 (s, 1H), 4.27 (q, J=7.0 Hz, 2H),2.19 (s, 3H), 1.28 (t, J=7.1 Hz, 3H); LC-MS: m/z 320.1 (M+H).

Step 3: Preparation of (S)-ethyl1-(2-(3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-nitrophenyl)-5-methyl-1H-pyrazole-3-carboxylate

To an ice cooled solution of2-(3-(ethoxycarbonyl)-5-methyl-1H-pyrazol-1-yl)-5-nitrobenzoic acid(25.0 g, 78.4 mmol) in DMF (250 mL) were added(S)-(1,2,3,4-tetrahydroisoquinolin-3-yl)methanol (31.0 g, 62.7 mmol),EDC.HCl (23.0 g, 117.5 mmol), HOBT (13.75 g, 101.9 mmol) followed bystirring at RT for 12 h. The reaction mixture was diluted with water,extracted with EtOAc (500 mL×2) twice. The combined organic layers werewashed with water (500 mL), brine (100 mL), dried over sodium sulphateand concentrated invacuo. The residue was purified on silica gel(100-200 mesh) to afford the ‘off white’ title compound 18 g (49%).Rf=0.45 (25% EtOAc in hexane); ¹H NMR (400 MHz, DMSO-d₆): δ 8.70-8.35(m, 2H), 8.15-7.98 (m, 1H), 7.25-6.95 (m, 4H), 6.82-6.58 (m, 1H),5.20-3.80 (m, 8H), 3.40-2.40 (m, 2H), 2.38-2.10 (m, 3H), 1.30-0.80 (m,3H); ES-MS: m/z 465.3 (M+H).

Step 4: Preparation of(S)-1-(2-(3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-nitrophenyl)-5-methyl-1H-pyrazole-3-carboxylicacid

To a solution of (S)-ethyl1-(2-(3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-nitrophenyl)-5-methyl-1H-pyrazole-3-carboxylate(18.0 g, 38.8 mmol) in THF (30 mL) and water (20 mL) was added lithiumhydroxide monohydrate (5.0 g, 116.8 mmol) followed by stirring at RT for6 h. The reaction mixture was concentrated invacuo, diluted with water(80 mL) and extracted with diethyl ether (100 mL). The aq. layer wascooled to 0° C., acidified up to pH ˜4 using 3N HCl and extracted withEtOAc (200 mL×2) twice. The combined organic layers were washed withwater (200 mL), brine (100 mL), dried over sodium sulphate andconcentrated invacuo. The residue was purified on silica gel (100-200mesh) to afford the title compound as a white colored solid 14 g (82%).Rf=0.25 (70% EtOAc in hexane); ¹H NMR (400 MHz, DMSO-d₆): δ 12.90-12.50(brs, 1H), 8.64-8.30 (m, 2H), 8.06-7.72 (m, 1H), 7.24-6.82 (m, 4H),6.80-6.42 (m, 1H), 5.20-3.80 (m, 6H), 3.40-2.40 (m, 2H), 2.40-2.10 (m,3H); ES-MS: m/z 437.2 (M+H).

Step 5: Preparation of(S)—N,N-dibutyl-1-(2-(3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-nitrophenyl)-5-methyl-1H-pyrazole-3-carboxamide

To a solution of(5)-1-(2-(3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-nitrophenyl)-5-methyl-1H-pyrazole-3-carboxylicacid (14.0 g, 32.1 mmol) in DMF (150 mL) were added dibutylamine (8.6mL, 48.2 mmol), EDC.HCl (9.2 g, 48.2 mmol), HOBT (5.63 g, 41.7 mmol)followed by stirring at RT for 12 h. The reaction mixture was dilutedwith water and extracted with EtOAc (300 mL×2) twice. The combinedorganic layers were washed with water (300 mL), brine (100 mL), driedover sodium sulphate and concentrated invacuo. The residue was purifiedon silica gel (100-200 mesh) to afford the title compound as and ‘offwhite’ solid 9 g (51%). Rf=0.47 (25% EtOAc in hexane); ¹H NMR (400 MHz,DMSO-d₆): δ 8.62-8.22 (m, 2H), 8.00-7.80 (m, 1H), 7.30-6.84 (m, 4H),6.60-6.35 (m, 1H), 5.20-3.80 (m, 3H), 3.79-2.40 (m, 9H), 2.38-2.20 (m,3H), 1.60-0.50 (m, 14H); LC-MS: m/z 548.0 (M+H).

Step 6: Preparation of(S)-1-(4-amino-2-(3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)phenyl)-N,N-dibutyl-5-methyl-1H-pyrazole-3-carboxamide

To a solution of(S)—N,N-dibutyl-1-(2-(3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-nitrophenyl)-5-methyl-1H-pyrazole-3-carboxamide(1.1 g, 2.0 mmol) in EtOH (10 mL) was added 10% Pd—C (0.04 g) followedby stirring under H₂ balloon pressure at RT for 5 h. The reactionmixture was filtered through celite, the bed was washed with EtOH (20mL) and the filtrate was concentrated invacuo. The residue was purifiedon silica gel (100-200 mesh) to afford the title compound as an ‘offwhite’ solid 0.8 g (77%). Rf=0.33 (40% EtOAc in hexane); ¹H NMR (400MHz, DMSO-d₆): δ 7.30-6.82 (m, 5H), 6.80-6.15 (m, 3H), 5.72-5.50 (m, 2H,D₂O exchangeable), 5.20-4.78 (m, 1H, D₂O exchangeable), 4.90-4.00 (m,2H), 4.00-2.22 (m, 9H), 2.20-2.00 (m, 3H), 1.60-0.60 (m, 14H); LC-MS:m/z 518.3 (M+H); HPLC: 98.64% (RT=5.997 min., method B).

Intermediate B(S)-1-(4-amino-2-(3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)phenyl)-N,N-dibutyl-5-methyl-1H-pyrazole-3-carboxamide

Step 1: Preparation of(S)—N,N-dibutyl-1-(2-(3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-nitrophenyl)-5-methyl-1H-pyrazole-3-carboxamide

To a solution of(S)—N,N-dibutyl-1-(2-(3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-nitrophenyl)-5-methyl-1H-pyrazole-3-carboxamide(9.0 g, 16.4 mmol) in DCM (150 mL) were added TBDMS-chloride (2.98 g,19.7 mmol), imidazole (2.23 g, 32.9 mmol) followed by stirring at RT for12 h. The reaction mixture was diluted with water and extracted withEtOAc (300 mL×2), twice. The combined organic layers were washed withwater (300 mL), brine (100 mL), dried over sodium sulphate andconcentrated invacuo. The residue was purified on silica gel (100-200mesh) to afford the title compound as a liquid 7 g (64%). Rf=0.74 (25%EtOAc in hexane); ¹H NMR (400 MHz, DMSO-d₆): δ 8.60-8.20 (m, 2H),8.05-7.80 (m, 1H), 7.40-6.80 (m, 4H), 6.60-6.30 (m, 1H), 5.10-3.84 (m,4H), 3.82-2.40 (m, 7H), 2.40-2.20 (m, 3H), 1.60-0.55 (m, 23H),0.05-−0.40 (m, 6H); LC-MS: m/z 662.4 (M+H).

Step 2: Preparation of(S)-1-(4-amino-2-(3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)phenyl)-N,N-dibutyl-5-methyl-1H-pyrazole-3-carboxamide

To a solution of(S)—N,N-dibutyl-1-(2-(3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-nitrophenyl)-5-methyl-1H-pyrazole-3-carboxamide(3.0 g, 4.5 mmol) in EtOH (50 mL) was added 10% Pd—C (0.3 g) followed bystirring under H₂ balloon pressure at RT for 3 h. The reaction mixturewas filtered through celite, the bed was washed with EtOH (150 mL) andthe filtrate was concentrated invacuo. The residue was purified onsilica gel (100-200 mesh) to afford the title compound as an ‘off white’solid 2.0 g (69%). Rf=0.45 (25% EtOAc in hexane); ¹H NMR (400 MHz,DMSO-d₆): δ 7.40-6.82 (m, 5H), 6.80-6.20 (m, 3H), 5.74-5.50 (m, 2H),5.10-4.00 (m, 2H), 3.99-2.20 (m, 9H), 2.20-2.00 (m, 3H), 1.60-0.58 (m,23H), 0.00-0.04 (m, 6H); LC-MS: m/z 632.6 (M+H).

Intermediate C(5)-1-(4-bromo-2-(3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydro-isoquinoline-2-carbonyl)phenyl)-N,N-dibutyl-5-methyl-1H-pyrazole-3-carboxamide

Step 1: Preparation of 5-bromo-2-hydrazinylbenzoic acid hydrochloride

To a suspension of 2-amino-5-bromobenzoic acid (50.0 g, 231.5 mmol) inconc. HCl (250 mL) at −10° C. was added a solution of sodium nitrite(23.92 g, 347.2 mmol) in water (250 mL), slowly, followed by stirringfor 2 h. To this reaction mixture was slowly added a solution ofstannous chloride (130.50 g, 225.6 mmol) in conc. HCl (125 mL) withcontinued stirring at RT for 12 h. The reaction mixture was filtered,the residue washed with the minimum amount of water and vacuum dried toafford the title compound as and ‘off white’ solid 61 g (100%), whichwas used for next step without further purification. Rf=0.10 (ethylacetate); ¹H NMR (400 MHz, DMSO-d₆): δ 11.60-9.60 (brs, 3H), 7.93 (d,J=2.4 Hz, 1H), 7.72 (dd, J=2.4 & 8.8 Hz, 1H), 7.12 (d, J=8.8 Hz, 1H);ES-MS: m/z 229.1 (M−H).

Step 2: Preparation of5-bromo-2-(3-(ethoxycarbonyl)-5-methyl-1H-pyrazol-1-yl)benzoic acid

To a solution of 5-bromo-2-hydrazinylbenzoic acid hydrochloride (60.0 g,225.6 mmol) in AcOH (600 mL) was added ethyl-2,4-dioxovalerate (35.67 g,225.6 mmol) followed by refluxing for 3 h. AcOH was evaporated invacuoand the residue was co-distilled with toluene (100 mL), twice. Theresidue was dissolved in EtOAc and washed with water until the aq. layerbecame neutral. The organic layer was dried over sodium sulphate andconcentrated invacuo. The gummy material was triturated with diethylether to afford the title compound as a brown colored oil 80 g (100%),which was used without further purification for the next step. Rf=0.23(10% MeOH in DCM); ¹H NMR (400 MHz, DMSO-d₆): δ 13.80-12.80 (brs, 1H),8.08 (d, J=1.9 Hz, 1H), 7.96 (dd, J=2.4&8.3 Hz, 1H), 7.53 (d, J=8.3 Hz,1H), 6.70 (s, 1H), 4.26 (q, J=7.2 Hz, 2H), 2.13 (s, 3H), 1.28 (t, J=7.1Hz, 3H); ES-MS: m/z 353.1 (M+H).

Step 3: Preparation of (S)-ethyl1-(4-bromo-2-(3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)phenyl)-5-methyl-1H-pyrazole-3-carboxylate

To an ice cooled solution of5-bromo-2-(3-(ethoxycarbonyl)-5-methyl-1H-pyrazol-1-yl)benzoic acid(45.0 g, 127.8 mmol) in DCM (450 mL) were added(S)-(1,2,3,4-tetrahydroisoquinolin-3-yl)methanol (20.8 g, 102.27 mmol),HATU (72.7 g, 191.2 mmol), DIPEA (55.7 mL, 319.6 mmol) followed bystirring at RT for 12 h. The reaction mixture was diluted with DCM (750mL), washed with water (500 mL), brine (100 mL), dried over sodiumsulphate and concentrated invacuo. The residue was purified on silicagel (100-200 mesh) to afford the title compound as a liquid 50 g (79%).Rf=0.44 (55% EtOAc in hexane); ¹H NMR (400 MHz, DMSO-d₆): δ 8.00-7.40(m, 3H), 7.30-7.00 (m, 4H), 6.80-6.40 (m, 1H), 5.10-3.80 (m, 7H),3.50-2.40 (m, 3H), 2.40-2.10 (m, 3H), 1.30-1.00 (m, 3H); ES-MS: m/z498.2 (M+H).

Step 4: Preparation of(S)-1-(4-bromo-2-(3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)phenyl)-5-methyl-1H-pyrazole-3-carboxylicacid

To a solution of (S)-ethyl1-(4-bromo-2-(3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)phenyl)-5-methyl-1H-pyrazole-3-carboxylate(50.0 g, 100.6 mmol) in THF (80 mL) and water (20 mL) was added lithiumhydroxide monohydrate (21.1 g, 503.0 mmol) followed by stirring at RTfor 12 h. The reaction mixture was concentrated invacuo, diluted withwater (80 mL) and extracted with diethyl ether (100 mL). The aq. layerwas cooled to 0° C., acidified up to pH ˜4 using 3N HCl and extractedwith EtOAc (200 mL×2), twice. The combined organic layers were washedwith water (200 mL), brine (100 mL), dried over sodium sulphate andconcentrated invacuo. The residue was purified on silica gel (100-200mesh) to afford the title compound as a pale yellow solid, 40 g (crude).Rf=0.10 (30% EtOAc in hexane); ¹H NMR (400 MHz, DMSO-d₆): δ 12.80-11.80(m, 1H), 8.15-7.40 (m, 3H), 7.30-6.90 (m, 4H), 6.70-6.30 (m, 1H),5.10-3.70 (m, 5H), 3.50-2.40 (m, 3H), 2.38-2.10 (m, 3H); ES-MS: m/z470.5 (M+H).

Step 5: Preparation of(S)-1-(4-bromo-2-(3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)phenyl)-N,N-dibutyl-5-methyl-1H-pyrazole-3-carboxamide

To a solution of(S)-1-(4-bromo-2-(3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)phenyl)-5-methyl-1H-pyrazole-3-carboxylicacid (10.0 g, 21.3 mmol) in DMF (100 mL) were added dibutylamine (3.7mL, 21.3 mmol), EDC.HCl (6.1 g, 31.9 mmol), HOBT (3.3 g, 21.3 mmol)followed by stirring at RT for 12 h. The reaction mixture was dilutedwith water, extracted with EtOAc (100 mL×2), twice. The combined organiclayers were washed with water (100 mL), brine (50 mL), dried over sodiumsulphate and concentrated invacuo. The residue was purified on silicagel (100-200 mesh) to afford the title compound as a hygroscopic solid,4.5 g (36%). Rf=0.44 (55% EtOAc in hexane); ¹H NMR (400 MHz, DMSO-d₆): δ8.00-7.40 (m, 3H), 7.30-6.80 (m, 4H), 6.50-6.20 (m, 1H), 5.10-4.00 (m,3H), 4.00-2.40 (m, 9H), 2.40-2.00 (m, 3H), 1.60-0.50 (m, 14H); LC-MS:m/z 581.4 (M+H); HPLC: 90.74% (RT=9.216 min., method C).

Step 6: Preparation of(S)-1-(4-bromo-2-(3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydro-isoquinoline-2-carbonyl)phenyl)-N,N-dibutyl-5-methyl-1H-pyrazole-3-carboxamide

To a solution of(S)-1-(4-bromo-2-(3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)phenyl)-N,N-dibutyl-5-methyl-1H-pyrazole-3-carboxamide(3.0 g, 5.2 mmol) in DCM (30 mL) were added TBDMS-chloride (0.93 g, 6.2mmol), imidazole (0.70 g, 10.3 mmol) followed by stirring at RT for 5 h.The reaction mixture was diluted with water, extracted with EtOAc (100mL×2), twice. The combined organic layers were washed with water (100mL), brine (50 mL), dried over sodium sulphate and concentrated invacuo.The residue was purified on silica gel (100-200 mesh) to afford thetitle compound as a liquid 3.0 g (83%). Rf=0.66 (50% EtOAc in hexane);¹H NMR (400 MHz, DMSO-d₆): δ 8.00-7.40 (m, 3H), 7.30-6.60 (m, 4H),6.55-6.20 (m, 1H), 5.00-4.10 (m, 2H), 4.10-2.40 (m, 6H), 2.40-2.00 (m,6H), 1.60-0.50 (m, 23H), 0.20-−0.40 (m, 6H); LC-MS: m/z 695.3 (M+H).

Intermediate D4-Chloro-1-[4-hydroxy-2-((S)-3-hydroxymethyl-3,4-dihydro-1H-isoquinoline-2-carbonyl)phenyl]-5-methyl-1H-pyrazole-3-carboxylicacid dibutylamide

Step 1: Preparation of 4-Chloro-5-methyl-1H-pyrazole-3-carboxylic acidethyl ester

A mixture of 5-methyl-1H-pyrazole-3-carboxylic acid ethyl ester (4.45 g,28.9 mmol) and N-chlorosuccinimide (5.01 g, 37.5 mmol) indimethylformamide (60 mL) was stirred for 24 hours at ambienttemperature. The reaction was then concentrated down and purified byeluting through a silica gel column with a 0 to 100% ethylacetate/heptane gradient to afford the title compound (5.2 g, 96% yield)as a white solid. MS (ESI) [m/e, (M+H)⁺]=189.3. ¹H NMR (400 MHz,chloroform-d) δ ppm 9.39 (br. s., 1H), 4.44 (q, J=7.1 Hz, 2H), 2.35 (s,3H), 1.43 (t, J=7.1 Hz, 3H).

Step 2: Preparation of 4-Chloro-5-methyl-1H-pyrazole-3-carboxylic aciddibutylamide

To a stirred solution of dibutylamine (13 mL, 76 mmol) indichloromethane (250 mL) under nitrogen atmosphere was addedtrimethylaluminium (38 mL, 2M in toluene, 76 mmol). The mixture wasstirred at ambient temperature for 30 minutes.(4-Chloro-5-methyl-1H-pyrazole-3-carboxylic acid ethyl ester (4.8 g, 25mmol) in dichloromethane (30 mL) was added dropwise to the mixture. Thereaction was stirred for 12 hours under a nitrogen atmosphere. Themixture was poured into saturated Rochelle salt solution slowly andstirred at ambient temperature for 2 hours. The organic layer wascollected. The aqueous layer was extracted with dichloromethane andcombined with the organic layer. The organic phase was brine-washed,dried over sodium sulfate, filtered, concentrated down, and dried. Theresidue was purified by eluting through a silica gel column with a 0 to100% ethyl acetate/heptane gradient to afford the title compound (4.7 g,68% yield) as a clear oil. MS (ESI) [m/e, (M+H)⁺]=272.4. ¹H NMR (400MHz, chloroform-d) δ ppm 3.50 (t, J=7.5 Hz, 2H), 3.37 (t, J=7.5 Hz, 2H),2.28 (s, 3H), 1.64 (quin, J=7.5 Hz, 2H), 1.44-1.55 (m, 2H), 1.30-1.43(m, 2H), 1.10-1.22 (m, 2H), 0.97 (t, J=8.0 Hz, 3H), 0.82 (t, J=7.3 Hz,3H).

Step 3: Preparation of4-Chloro-1-(2-cyano-4-methoxy-phenyl)-5-methyl-1H-pyrazole-3-carboxylicacid dibutylamide

A mixture of 4-chloro-5-methyl-1H-pyrazole-3-carboxylic aciddibutylamide (1.5 g, 5.5 mmol), 2-fluoro-5-methoxybenzonitrile (1.1 g,7.2 mmol), and cesium carbonate (1.8 g, 5.5 mmol) in dimethylformamide(5 mL) was microwaved at 130° C. for 30 minutes. The solvent was removedin vacuo. The crude material was eluted through a silica gel column witha 0 to 70% ethyl acetate/heptane gradient to afford the title compound(1.3 g, 59% yield) as a white solid. MS (ESI) [m/e, (M+H)⁺]=403.5. ¹HNMR (400 MHz, chloroform-d) δ ppm 7.32 (d, J=8.5 Hz, 1H), 7.18-7.22 (m,1H), 7.12-7.18 (m, 1H), 3.84 (s, 3H), 3.41-3.51 (m, 2H), 3.32-3.41 (m,2H), 2.15 (s, 3H), 1.54-1.66 (m, 2H), 1.48 (qd, J=7.7, 7.5 Hz, 2H),1.26-1.40 (m, 2H), 1.17 (ddd, J=14.9, 7.4, 7.3 Hz, 2H), 0.89 (t, J=7.3Hz, 3H), 0.77 (t, J=7.3 Hz, 3H).

Step 4: Preparation of2-(4-Chloro-3-dibutylcarbamoyl-5-methyl-pyrazol-1-yl)-5-methoxy-benzoicacid

A mixture of4-Chloro-1-(2-cyano-4-methoxy-phenyl)-5-methyl-1H-pyrazole-3-carboxylicacid dibutylamide (1.3 g, 3.2 mmol) and potassium hydroxide (1.2 g, 21mmol) in ethanol (5 mL) and water (5 mL) was microwaved at 150° C. for90 minutes. The pH was adjusted to about 5 with aqueous HCl solution (15N). The solvent was removed in vacuo. The crude material was elutedthrough a silica gel column with a 10 to 100% ethyl acetate/heptane andthen 1 to 20% methanol/methylene chloride gradient to afford the titlecompound (0.53 g, 39% yield) as a white solid. MS (ESI) [m/e,(M+H)⁺]=422.4.

Step 5: Preparation of4-Chloro-1-[2-((S)-3-hydroxymethyl-3,4-dihydro-1H-isoquinoline-2-carbonyl)-4-methoxy-phenyl]-5-methyl-1H-pyrazole-3-carboxylicacid dibutylamide

To a stirred solution of2-(4-chloro-3-dibutylcarbamoyl-5-methyl-pyrazol-1-yl)-5-methoxy-benzoicacid (0.35 g, 0.82 mmol) and(S)-(1,2,3,4-tetrahydroisoquinolin-3-yl)methanol (0.13 g, 0.82 mmol) indichloromethane (8 mL) under nitrogen atmosphere was added1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.16 g, 0.82 mmol) andhydroxybenzotriazole (0.13 g, 0.82 mmol). The mixture was stirred atambient temperature for 5 minutes. Triethylamine (0.34 mL, 2.5 mmol) wasadded to the mixture. The reaction was stirred for 60 hours at ambienttemperature. The mixture was washed with water and purified by elutingthrough a silica gel column with a 10 to 100% ethyl acetate/heptanegradient to afford the title compound (21 mg, 4.5% yield). MS (ESI)[m/e, (M+H)⁺]=567.3. ¹H NMR (400 MHz, chloroform-d) δ ppm 6.75-7.36 (m,7H), 4.13-5.41 (m, 4H), 3.80-3.96 (m, 3H), 2.51-3.71 (m, 8H), 2.17-2.32(m, 3H), 1.48-1.68 (m, 4H), 1.19-1.44 (m, 4H), 0.70-0.97 (m, 6H).

Step 6:4-Chloro-1-[4-hydroxy-2-((S)-3-hydroxymethyl-3,4-dihydro-1H-isoquinoline-2-carbonyl)-phenyl]-5-methyl-1H-pyrazole-3-carboxylicacid dibutylamide

A mixture of4-chloro-1-[2-((S)-3-hydroxymethyl-3,4-dihydro-1H-isoquinoline-2-carbonyl)-4-methoxy-phenyl]-5-methyl-1H-pyrazole-3-carboxylicacid dibutylamide (40 mg, 0.071 mmol) and aluminium chloride (75 mg,0.56 mmol) was stirred in ethanethiol (0.052 mL, 0.71 mmol) anddichloromethane (0.5 mL) for 12 hours at ambient temperature under anitrogen atmosphere. The mixture was added with water and extracted with1:4 methanol:methylene chloride. The organic layer was removed in vacuo.After eluting through a short pad of silica gel column and C18 column,the crude material was purified by HPLC to afford the title compound (9mg, 33% yield). MS (ESI) [m/e, (M+H)⁺]=553.5. ¹H NMR (400 MHz, DMSO-d₆)δ ppm 6.66-7.50 (m, 7H), 2.03-5.08 (m, 14H), 0.53-1.62 (m, 14H).

Intermediate E1-[5-Hydroxy-2-((S)-3-hydroxymethyl-3,4-dihydro-1H-isoquinoline-2-carbonyl)-phenyl]-5-methyl-1H-pyrazole-3-carboxylicacid dibutylamide

Step 1: Preparation of 5-Methyl-1H-pyrazole-3-carboxylic aciddibutylamide

Following Preparation of Intermediate D/Step 2, the title compound (6 g,65%) was prepared from 5-methyl-1H-pyrazole-3-carboxylic acid ethylester. MS (ESI) [m/e, (M+H)⁺]=238.1. ¹H NMR (400 MHz, chloroform-d) δppm 6.29 (s, 1H), 3.59 (t, J=7.5 Hz, 2H), 3.38-3.51 (m, 2H), 2.32 (s,3H), 1.47-1.74 (m, 4H), 1.17-1.47 (m, 4H), 0.76-1.03 (m, 6H).

Step 2: Step 2:2-(3-Dibutylcarbamoyl-5-methyl-pyrazol-1-yl)-4-methoxy-benzoic acid

A mixture of 5-methyl-1H-pyrazole-3-carboxylic acid dibutylamide (0.85g, 3.6 mmol), 2-iodo-4-methoxybenzoic acid (1.0 g, 3.6 mmol), copperiodide (0.14 g, 0.72 mmol), cesium carbonate (1.2 g, 3.6 mmol), andtrans-dimethylaminecyclohexane (0.23 mL, 1.44 mmol) in dioxane (5 mL)was microwaved at 120° C. for 15 minutes. Diluted with ethyl acetate,the crude material was eluted through a silica gel column with a 0 to100% ethyl acetate/heptane and then 0 to 20% methanol/methylene chloridegradient to afford the title compound (0.43 g, 31% yield). MS (ESI)[m/e, (M+H)⁺]=388.5.

Step 3:1-[2-((S)-3-Hydroxymethyl-3,4-dihydro-1H-isoquinoline-2-carbonyl)-5-methoxy-phenyl]-5-methyl-1H-pyrazole-3-carboxylicacid dibutylamide

Following Preparation of Intermediate D/Step 5, the title compound (610mg, 29%) was prepared from2-(3-dibutylcarbamoyl-5-methyl-pyrazol-1-yl)-4-methoxy-benzoic acid. MS(ESI) [m/e, (M+H)⁺]=533.3. ¹H NMR (400 MHz, chloroform-d) δ ppm6.81-7.33 (m, 7H), 5.95-6.40 (m, 1H), 4.28-5.62 (m, 4H), 3.83-3.96 (m,3H), 2.66-3.81 (m, 7H), 2.01-2.38 (m, 3H), 1.10-1.65 (m, 8H), 0.76-1.01(m, 6H).

Example 1N,N-dibutyl-1-(2-(3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-(phenylsulfonamido)phenyl)-5-methyl-1H-pyrazole-3-carboxamide

To a ice-cooled solution of(S)-1-(4-amino-2-(3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)phenyl)-N,N-dibutyl-5-methyl-1H-pyrazole-3-carboxamide(intermediate B, 0.20 g, 0.316 mmol) in DCM (2 mL) was added Et₃N (0.05mL, 0.47 mmol) and benzenesulfonyl chloride (0.06 g, 0.38 mmol) followedby stirring at RT for 16 h. The reaction mixture was diluted with water(20 mL) and extracted with EtOAc (100 mL). The organic layer was washedwith brine (50 mL), dried over sodium sulphate and concentrated invacuo.The residue was purified on silica gel (100-200 mesh) to afford(S)—N,N-dibutyl-1-(2-(3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-(phenylsulfonamido)phenyl)-5-methyl-1H-pyrazole-3-carboxamideas a mixture of mono and bis sulfonamides, 0.21 g (crude). The crudereaction mass was used in the next reaction without furtherpurification. Rf=0.75 (50% EtOAc in hexane); ES-MS: m/z 772.6 (M+H).

A mixture of(S)—N,N-dibutyl-1-(2-(3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-(phenylsulfonamido)phenyl)-5-methyl-1H-pyrazole-3-carboxamideand(S)—N,N-dibutyl-1-(2-(3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-(N-(phenylsulfonyl)phenylsulfonamido)phenyl)-5-methyl-1H-pyrazole-3-carboxamide(0.21 g, 0.272 mmol) in THF (2.1 mL) was treated with 3N HCl (0.62 mL)at RT for 10 min. The reaction mixture was diluted with water (20 mL),the pH was adjusted to about 8 using aq. NaHCO₃ solution and extractedwith EtOAc (100 mL). The organic layer was washed with water (50 mL),brine (50 mL), dried over sodium sulphate and concentrated invacuo. Theresidue was purified on silica gel (100-200 mesh) to afford the titlecompound as a hygroscopic solid, 0.025 g (14%). Rf=0.38 (5% MeOH inDCM); ¹H NMR (400 MHz, DMSO-d₆): δ 10.85-10.70 (m, 1H, D₂Oexchangeable), 7.78-6.60 (m, 12H), 6.50-6.20 (m, 1H), 5.00-3.60 (m, 4H),3.60-2.40 (m, 8H), 2.25-2.00 (m, 3H), 1.60-0.50 (m, 14H); LC-MS: m/z658.4 (M+H), HPLC: 95.08% (RT=9.403 min., method I).

By repeating the procedures described in the above example, usingappropriate starting materials and HPLC methods, the following compoundsof Formula I, as identified in Table 1, are obtained.

TABLE 1 BCL2_SPR BCL2_SPR MW HPLC IC50 IC50 (ESI, RT BAD BAK ExampleStructure (M + H)⁺) (min) (nM) (nM) 1

658.4 9.403 629.08- 755.79 474.19- 563.56 2

596.4 8.419 367.13- 387.28 259.8- 329.63 3

623.8 8.614 125.84- 127.28 87.83- 93.04 4

804.0 2.641 790.78- 808 661.04- 689.76 5

699.9 3.162 616.04- 602.45 413.61- 410.87 6

777.2 1.819 543.48- 582.07 448.2- 482.14 7

815.6 3.115 642.6- 633.86 475.61- 471.67 8

738.9 2.981 869.16- 802.56 685.09- 643.43 9

768.4 3.357 >16000 >16000 9031.86- 6839.48 10

764.0 3.339 >16000 >16000 3022.25- 3677.07 11

754.9 3.193 271.76- 320.45 263.29- 243.02 12

764.0 3.355 6743.92- 6516.76 4876.33- 4850.83 13

802.8 3.210 6815.26- 7741.56 4629.28- 3792.69 14

692.3 2.955 726.32- 702.68 434.59- 682.75 15

764.8 3.290 2112.18- 2237.41 1721.13- 1943.36 16

661.8 2.918 635.94- 643.97 477.67- 472.48 17

710.3 3.064 976.89- 937.42 828.41- 870.71 18

705.8 3.175 542.02- 627.35 491.04- 422.3 19

705.9 2.943 20

737.9 2.846 873.96- 836.42 727.16- 786.91 21

710.9 3.080 1362.99- 1350.59 1105.04- 1001.31 22

740.8 3.180 1747- 1559.39 1853.03- 2108.91 23

624.8 2.092 2412.41- 2363.56 1766.36- 1705.19 24

689.9 3.057 621.59- 681.94 441.19- 491.5 25

749.3 3.113 418.35- 435.82 252.06- 256.94 26

726.6 2.948 493.04- 494.05 375.53- 365.33 27

675.8 2.761 465.84- 442.47 304.28- 325.62 28

715.9 3.123 454.68- 440.7 310.27- 311.45 29

735.9 3.047 712.16- 706.56 508.71- 549.45 30

831.5 3.064 2664.8- 2588.57 2278.5- 2506.72 31

767.93 3.249 4796.85- 6028.18 3247.78- 3818.27 32

715.9 3.249 592.6- 670.37 529.43- 493.71 33

737.9 2.876 5311.59- 5287.81 3505.41- 3612.22 34

739.9 3.249 845.42- 840.85 636.43- 646.48 35

699.8 2.891 250.64- 292.9 258.27- 256.04 36

745.9 3.196 1132.89- 1019.53 787.69- 880.62 37

764.0 2.329 >16000 >16000 >16000 >16000 38

772.7 3.128 284.07- 253.57 334.36- 321.5 39

751.9 3.219 631.4- 633.01 577.11- 554.26 40

682.8 3.177 699.01- 646.14 621.62- 502.84 41

716.9 3.286 682.21- 638.78 479.16- 582.13 42

702.8 3.212 263.57- 277.19 221.74- 287.65 43

732.0 3.047 820.9- 793.06 767.45- 685.1 44

724.9 3.103 863.4- 905.85 540.69- 503.5 45

721.9 3.235 474.69- 485.27 286.41- 299.4 46

671.3 3.03 646.28- 685.79 480.92- 468.54

BCL-2 binding can be determined using a variety of known methods. Onesuch assay is a sensitive and quantitative in vitro binding assay usingfluorescence polarization (FP) described by Wang, J.-L.; Zhang, Z-J.;Choksi, S.; Sjam. S.; Lu, Z.; Croce, C. M.; Alnemri, E. S.; Komgold, R.;Huang, Z. Cell permeable BCL-2 binding peptides: a chemical approach toapoptosis induction in tumor cells. Cancer Res. 2000, 60, 1498-1502).

Methods for Determining IC50s

The present method includes utility of a Surface plasmon resonance(SPR)-based biosensor (Biacore™′ GE Healthcare, Uppsala, Sweden) tocharacterize BCL-2 inhibitors.

Biacore™ utilizes the phenomenon of surface plasmon resonance (SPR) todetect and measure binding interactions. In a typical Biacoreexperiment, one of the interacting molecules (ligand) is immobilized ona flexible dextran matrix while the interacting partner (analyte) isallowed to flow across that surface. A binding interaction results in anincrease in mass on the sensor surface and a corresponding direct changein the refractive index of the medium in the vicinity of the sensorsurface. Changes in refractive index or signal are recorded in resonanceunits (R.U.) Signal changes due to association and dissociation ofcomplexes are monitored in a non-invasive manner, continuously and inreal-time, the results of which are reported in the form of asensorgram.

The SPR assay is configured to examine solution inhibition of BCL-2binding to peptide derivatized sensor surfaces to generate IC50 valuesas a measure of inhibitor potency.

Solution Inhibition Assay Format:

Biacore™ A100 (GE Healthcare, Uppsala, Sweden) was used to conduct allexperiments reported herein. Sensor surface preparation and allinteraction analyses experiments were performed at 25° C. Reagents werepurchased from GE Healthcare. Running buffer containing 10 mM Hepes,pH7.4, 150 mM sodium chloride, 1.25 mM Dithiothreitol, 3% Dimethylsulfoxide and 0.05% polysorbate 20 were utilized throughout allanalyses.

Biotinylated BAK, BAD and NOXA peptides were diluted to 10 nM in runningbuffer and captured onto a sensor surface pre-derivatized withstreptavidin (sensor chip SA) to peptide surface densities in the range50-100 R.U. Peptide captured surfaces were blocked with 500 μMPEO₂-Biotin. A blank detection spot in each flowcell was similarlyblocked with PEO₂-biotin and served as a reference spot in thecompetition assay.

Interaction analyses were performed by first equilibrating each samplewithin a 6 point three fold compound dilution series in the range 1604to 0.004 nM with 56 nM BCL-2 for one hour during instrument start-upprocedures. Protein compound mixtures were then injected over eachpeptide surface in parallel for 60 seconds at a flow-rate of 30 μL/min.56 nM BCL-2 control samples were also prepared and run at regularintervals during the assay. Surface regeneration was performed at theend of each analysis cycle by two 30 second injections of 10 mM Glycine,pH 2.5, 1M Sodium Chloride, 0.05% polysorbate 20. Samples and controlcompound samples were run in duplicate and controls are also run atregular intervals during the assay to monitor surface and assayperformance.

Data analyses are carried out using Biacore™ A100 evaluation softwarev1.1 to validate assay quality. Binding level report points were usedrelative to BCL-2 control samples to calculate % inhibition values foreach compound protein mixture. These data are then plotted versuscompound concentration and analyzed in Tibco® Spotfire® v2.1 vialogistic regression to calculate IC₅₀ values for each compound. Therange of data shown in table 1 represents the high and low IC₅₀ valuesobtained from multiple experiments.

Caspase Activation Assay Method

In cancer cell lines that depend on BCL2 for survival, such as theCaki-2 kidney clear cell carcinoma cell line, inhibition of BCL2 inducesapoptosis characterized by activation of caspases. Compounds of theinvention are tested for ability to induce caspase activation in theCaki-2 cell line as follows. On day one, 2500 Caki-2 cells are platedinto 384 well tissue culture plates. On day two, cells are treated witha dose range of a compound of the invention in Opti-MEM media(Invtrogen) containing 1% fetal bovine serum for four hours. At fourhours post-treatment relative levels of caspase activation, relative tobaseline levels in vehicle treated cells, are assessed using theCaspase-glo reagent from Promega.

Cell Proliferation Assay Method

Compounds of the invention are tested for their ability to impact cellproliferation and/or survival in the Caki-2 cell line as follows. On dayone, 2500 Caki-2 cells are plated into 384 well tissue culture plates.On day two, cells are treated with a dose range of a compound of theinvention in Opti-MEM media (Invitrogen) containing 1% fetal bovineserum for 24 hours. At 24 hours post-treatment, cell viability, relativeto vehicle treated cells, is assessed using the ATPLite reagent fromPerkin Elmer.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

The invention claimed is:
 1. A compound of formula Ia:

in which: R₁ is hydrogen; R₂ is hydrogen; R₄ is hydroxy; and R₅ isselected from C₁₋₆alkyl, C₂₋₆alkenyl, cyclopropyl,imidazo[1,2-a]pyrimidinyl, 2-oxo-4-phenylpiperazin-1-yl,4-(2-chlorobenzyl)-3-oxopiperazin-1-yl, imidazo[1,2-a]pyridinyl,2-oxo-1,2,3,6-tetrahydropyrimidinyl,naphtho[2,1-d][1,2,3]oxadiazol-5-yl,(7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methyl,1H-pyrrolo[2,3-b]pyridinyl, 1H-pyrazolo[3,4-b]pyridinyl,benzo[c][1,2,5]thiadiazolyl, imidazo[2,1-b]thiazolyl,2,3-dihydrobenzo[b][1,4]dioxin-2-yl, naphtho[2,3-d][1,3]dioxol-2-yl,3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl, 1,2,4-oxadiazolyl,4-oxo-4,5,6,7-tetrahydrobenzofuranyl, 5-oxopyrrolidin-3-yl,benzo[d]isoxazolyl, 2,3-dihydrobenzofuran-3-yl, chroman-8-yl,3-oxo-3H-pyrazolyl, benzo[b]thiophenyl, benzo[b]furanyl,6-oxo-1,6-dihydropyridazinyl, 2-oxo-1,2-dihydropyridinyl,2-oxo-1,2,5,6,7,8-hexahydroquinolinyl,4-oxo-1,4-dihydro-1,8-naphthyridinyl, 4-oxo-4H-pyrano[2,3-b]pyridinyl,10,10-dioxido-9-oxo-9H-thioxanthen-3-yl, 1,2,5-oxadiazolyl,dimethyl-amino, benzo[c][1,2,5]oxadiazolyl, isoindoline-1,3-dionyl,phenyl, benzyl, quinolinyl, isoquinolinyl, phenoxy, benzoxy, phenylthio,pyrrolyl, quinolin-8-yloxy, phenyl-sulfonyl, furanyl, thiazolyl,oxazolyl, isoxazolyl, thienyl, pyridinyl, pyrimidinyl, pyrrolidinyl,imidazolidine-2,4-dionyl, pyrrolidinonyl, piperidinyl, piperazinyl,pyrazinyl, pyrazolyl, morpholino, oxomorpholino, indolyl,benzo[d][1,2,3]triazol and oxopiperazinyl; wherein said C₁₋₆ alkyl, C₂₋₆alkenyl, cyclopropyl, imidazo[1,2-a]pyrimidinyl,imidazo[1,2-a]pyridinyl, 1H-pyrrolo[2,3-b]pyridinyl, benzo[d]isoxazolyl,1H-pyrazolo[3,4-b]pyridinyl, 1,2,4-oxadiazolyl,benzo[c][1,2,5]thiadiazolyl, imidazo[2,1-b]thiazolyl,4-oxo-4,5,6,7-tetrahydrobenzofuranyl,2-oxo-1,2,3,6-tetrahydropyrimidinyl,2,3-dihydrobenzo[b][1,4]dioxin-2-yl, naphtho[2,3-d][1,3]dioxol-2-yl,3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl, 2,3-dihydrobenzofuran-3-yl,chroman-8-yl, 3-oxo-3H-pyrazolyl, 6-oxo-1,6-dihydropyridazinyl,2-oxo-1,2-dihydropyridinyl, benzo[b]thiophenyl, 1,2,5-oxadiazolyl,benzyl, benzo[c][1,2,5]oxadiazolyl, isoindoline-1,3-dionyl,benzo[b]furanyl, 2-oxo-1,2,5,6,7,8-hexahydroquinolinyl,4-oxo-1,4-dihydro-1,8-naphthyridinyl, 4-oxo-4H-pyrano[2,3-b]pyridinyl,10,10-dioxido-9-oxo-9H-thioxanthen-3-yl, 5-oxopyrrolidin-3-yl, phenyl,quinolinyl, pyrimidinyl, isoquinolinyl, phenoxy, benzoxy,phenoxy-methyl, phenylthio, pyrrolyl, quinolin-8-yloxy, phenyl-sulfonyl,furanyl, thiazolyl, oxazolyl, isoxazolyl, thienyl, pyridinyl,pyrrolidinyl, pyrrolidinonyl, piperazinyl, piperidinyl, pyrazinyl,pyrazolyl, morpholino, oxomorpholino, indolyl, benzo[d][1,2,3]triazol oroxopiperazinyl of R₅ is unsubstituted or substituted with 1 to 3 groupsindependently selected from halo, cyano, nitro, —NR₆R₇, C₁₋₄alkyl,halo-substituted-C₁₋₄alkyl, C₁₋₄ alkoxy, halo-substituted-C₁₋₄ alkoxy,halo-substituted-C₁₋₄ alkylthio, —C(O)OR₆, —C(O)R₆, —X₃OR₆,—X₃NR₆C(O)R₇, —NR₆S(O)₂R₇, —C(O)NR₆R₇, —S(O)₀₋₂R₆, —S(O)₀₋₂NR₆R₇,phenyl, pyridinyl, benzyl, morpholino, oxazolyl, naphthyl, pyrrolidinyl,piperidinyl, morpholino-methyl, 1,3-dioxoisoindolinyl,1,2,4-oxadiazolyl, pyrazolyl, indolyl, (1H-1,2,4-triazolyl)methyl,phenoxy and benzoxy; wherein R₆ and R₇ are independently selected fromhydrogen, C₁₋₄alkyl, C₃₋₈cycloalkyl, pyridinyl, pyrrolidinyl, phenyl,benzyl and naphthyl; wherein said phenyl, pyridinyl, pyrrolidinyl,benzyl, morpholino, morpholino-methyl, 1,2,4-oxadiazolyl, pyrazolyl,indolyl and benzoxy substituents of R₅ or said pyridinyl and phenyl ofR₆ or R₇ can be unsubstituted or further substituted with a groupselected from halo, nitro, amino-sulfonyl, C₁₋₄alkyl, C₁₋₄alkoxy andhalo-substituted-C₁₋₄alkyl; wherein X₃ is selected from a bond andC₁₋₄alkylene; or a pharmaceutically acceptable salt thereof.
 2. Thecompound of claim 1 or a pharmaceutically acceptable salt thereof,wherein the group

in formula (Ia) has the (S) stereochemistry.
 3. A compound selectedfrom:

or a pharmaceutically acceptable salt thereof.
 4. A compound of claim 1or a salt thereof in combination with one or more therapeutically activeagents.
 5. The compound of claim 1, wherein: R₅ is selected fromC₁₋₆alkyl, (7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methyl,benzo[b]thiophenyl, dimethyl-amino, benzo[c][1,2,5]oxadiazolyl, phenyl,benzyl, furanyl, oxazolyl, isoxazolyl, thienyl, pyrazolyl, and indolyl;wherein said C₁₋₆alkyl, benzo[b]thiophenyl, phenyl, benzyl, furanyl,oxazolyl, isoxazolyl, thienyl, pyrazolyl, and indolyl of R₅ isunsubstituted or substituted with 1 to 3 groups independently selectedfrom halo, cyano, nitro, C₁₋₄alkyl, —C(O)OR₆, —C(O)R₆, —X₃OR₆,—X₃NR₆C(O)R₇, —S(O)₀₋₂R₆, phenyl, oxazolyl, naphthyl,1,3-dioxoisoindolinyl, 1,2,4-oxadiazolyl, and pyrazolyl; wherein R₆ andR₇ are independently selected from hydrogen, C₁₋₄alkyl, and phenyl,wherein said phenyl, 1,2,4-oxadiazolyl, and pyrazolyl substituent of R₅or said phenyl of R₆ or R₇ can be unsubstituted or further substitutedwith a group selected from halo and C₁₋₄alkyl; wherein X₃ is selectedfrom a bond and C₁₋₄alkylene; or a pharmaceutically acceptable saltthereof.
 6. The compound of claim 1, wherein: R₅ is selected fromC₁₋₆alkyl, (7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methyl,benzo[b]thiophenyl, benzo[c][1,2,5]oxadiazolyl, phenyl, benzyl, furanyl,oxazolyl, isoxazolyl, thienyl, pyrazolyl, and indolyl; wherein saidC₁₋₆alkyl, benzo[b]thiophenyl, phenyl, benzyl, furanyl, oxazolyl,isoxazolyl, thienyl, pyrazolyl, and indolyl of R₅ is unsubstituted orsubstituted with 1 to 3 groups independently selected from halo, cyano,nitro, C₁₋₄alkyl, —C(O)OR₆, —C(O)R₆, —X₃OR₆, —X₃NR₆C(O)R₇, —S(O)₀₋₂R₆,phenyl, oxazolyl, naphthyl, 1,3-dioxoisoindolinyl, 1,2,4-oxadiazolyl,and pyrazolyl; wherein R₆ and R₇ are independently selected fromhydrogen, C₁₋₄alkyl, and phenyl, wherein said phenyl, 1,2,4-oxadiazolyl,and pyrazolyl substituent of R₅ or said phenyl of R₆ or R₇ can beunsubstituted or further substituted with a group selected from halo andC₁₋₄alkyl; wherein X₃ is selected from a bond and C₁₋₄alkylene; or apharmaceutically acceptable salt thereof.
 7. The compound of claim 1,wherein: R₅ is C₁₋₆alkyl; or a pharmaceutically acceptable salt thereof.8. The compound of claim 5, or a pharmaceutically acceptable saltthereof, wherein the group

in formula (Ia) has the (S) stereochemistry.
 9. The compound of claim 6,or a pharmaceutically acceptable salt thereof, wherein the group

in formula (Ia) has the (S) stereochemistry.
 10. The compound of claim7, or a pharmaceutically acceptable salt thereof, wherein the group

in formula (Ia) has the (S) stereochemistry.
 11. A pharmaceuticalcomposition comprising a compound of claim 1 or a pharmaceuticallyacceptable salt thereof, and at least one pharmaceutically acceptablecarrier.
 12. A method of treating cancer comprising administering acompound of claim 1, or a pharmaceutically acceptable salt thereof, to aperson in need of such treatment in an effective amount for theprophylactic or therapeutic treatment of cancer wherein the cancer isselected from prostate, hormone resistant prostate, breast, non-smallcell lung, small cell lung, colorectal, melanoma, head, neck andpancreatic cancer.